Humanized anti-cd40 antibodies conjugated to therapeutic agents

ABSTRACT

Provided are humanized anti-CD40 antibodies and antigen-binding fragments and methods for treating disease characterized by expression of CD40 antigen.

CONTINUITY

This application is a continuation of U.S. application Ser. No.13/622,517, filed Sep. 19, 2012; which is a divisional of U.S.application Ser. No. 11/913,305, filed Oct. 24, 2008, now U.S. Pat. No.8,303,955; which is the National Stage of International Application No.PCT/US2006/020688, filed May 26, 2006; all three of which claim thebenefit of U.S. Provisional Application No. 60/684,853, filed May 26,2005; the disclosures of all four applications are incorporated byreference herein in their entirety.

REFERENCE TO A SEQUENCE LISTING

This application includes a sequence listing as a text file, named0040-00213US_ST25.txt created Jun. 24, 2013, the content of which isincorporated herein by reference in its entirety.

BACKGROUND

This invention generally relates to humanized anti-CD40 antibodies fordiagnostic and therapeutic use. More specifically, humanized anti-CD40antibodies and methods of use for the treatment of various diseases ordisorders characterized by cells expressing CD40 are disclosed.Pharmaceutical compositions and articles of manufacture such as kitscomprising the humanized anti-CD40 antibody are also disclosed.

CD40 is a type I integral membrane glycoprotein and a member of thetumor necrosis factor (TNF) receptor superfamily. CD40 is expressed on avariety of cell types including normal and neoplastic B cells,interdigitating cells, basal epithelial cells and carcinomas. It is alsopresent on monocytes, macrophages, some endothelial cells, andfollicular dendritic cells. CD40 is expressed early in B cell ontogeny,appearing on B cell precursors subsequent to the appearance of CD10 andCD19, but prior to expression of CD21, CD23, CD24, and appearance ofsurface immunoglobulin M (sIgM) (Uckun et al., 1990, Blood 15:2449).Although early reports indicated that CD40 was lost upon terminaldifferentiation of B cells into plasma cells, CD40 has been detected ontonsil and bone marrow-derived plasma cells (Pellat-Decounynck et al.,1994, Blood 84:2597).

The interaction of CD40 with its ligand and counter-receptor, CD40L(also referred to as CD154, gp39, and TRAP), induces both humoral andcell-mediated immune responses. CD40L is a transmembrane proteinexpressed predominantly on activated lymphocytes. CD4⁺ T cells. Likeother proteins in the TNF family, the structure of CD40L is that of anoncovalent trimer. CD40-mediated signaling appears to be required for Bcell proliferation, immunoglobulin (Ig) isotype switching, germinalcenter formulation, and memory B cell commitment in response to Tcell-dependent antigen. CD40 binding of CD40L results in CD40multimerization, the generation of activation signals for antigenpresenting cells such as dendritic cells, monocytes, and B cells, andthe generation of growth and differentiation signals forcytokine-activated fibroblasts and epithelial cells. While the signalingpathways through which CD40 molecules function in cell differentiationhave not been completely elucidated, CD40 signals are transduced fromthe multimerized receptor via recruitment of a series of TNF receptorassociated factors (“TRAFs”) (Kehry, 1996, J. Immumol. 156:2345-2348).Subsets of TRAFs interact differentially with TNF receptor familymembers, including CD40, providing stimuli to a wide variety ofdownstream pathways. TRAF1 and TRAF2 are implicated in the modulation ofapoptosis (Speiser et al., 1997, J. Exp. Med. 185:1777-1783; Yeh et al.,1997, Immunity 7:715-725). TRAFs 2, 5, and 6 participate inproliferation and activation events. In normal B cells, binding of CD40to CD40L recruits TRAF2 and TRAF3 to the receptor complex and inducesdown regulation of other TRAF's (Kuhne et al., 1997, J. Exp. Med.186:337-342).

Apoptosis and CD40-mediated signaling are closely linked during B celldevelopment and differentiation. A primary function of apoptosis in Bcells is the clonal deletion of immature B cells, which is thought toresult from extensive cross-linking of surface Ig in immature B cells.The fate of mature B cells is also modulated by a combination ofsignaling via surface Ig and signals derived form activated T cells,presumably mediated by CD40L molecules. A combination of signals fromsurface Ig and CD40 can override the apoptotic pathway and maintaingerminal center B cell survival. This rescue from apoptosis in germinalcenters is critical for the development of affinity antibody-producingmemory B cells.

In both T and B cell malignancies, antitumor effects (growth arrest withor without apoptosis) often result when malignant cells are exposed tostimuli that lead to activation of normal lymphocytes. Thisactivation-induced growth arrest has been observed with signals througheither antigen receptors or costimulatory receptors (Ashwell et al.,1987, Science 237:61; Bridges et al., 1987, J. Immumol. 139:4242; Pageand Defranco, 1988 J. Immunol. 140:3717; and Beckwith et al., 1990, J.Natl. Cancer Inst. 82:501). CD40 stimulation by either anti-CD40antibody or soluble CD40L directly inhibits B cell lymphoma growth(Funakoshi et al., 1994, Blood 83:2787-2784).

Several murine monoclonal antibodies (mAbs) directed against CD40 havebeen described (Katira et al. 1995, “CD40 Workshop Panel Report”; In:Leukocyte Typing V, Schlossman et al., (eds) 1995, 1:547-550). Forexample, two mAbs, CD40.7 (M2) and CD40.8 (M3), were shown to inhibitthe binding of CD40 to CD40L (Fanslow et al., 1995, In: Leukocyte TypingV, Schlossman et al., (eds) 1995, 1:555-556). CD40 stimulation by mAbsM2 and M3 inhibited growth of several human B-cell lymphomas and inducedregression of established tumors in vivo (Funakoshi et al., 1994, Blood83:2787-2794; Funakoshi et al., 1996, J. Immunol. 19:93-101). U.S. Pat.No. 5,182,368 discloses an anti-CD40 murine mAb, G28-5, which canaugment B cell proliferation. A single chain immunotoxin based thesingle-chain Fv region of G28-5 selectively killed human CD40-expressinghematologic malignant cell lines in vitro (Francisco et al., 1997, JBiol. Chem. 39:24165-24169). However, G28-5 does not enhance activationof B cells in the presence of CD40L and does not potentiate the bindingof CD40 and CD40L. U.S. Pat. No. 6,838,261 (and related U.S. Pat. Nos.6,946,129 and 6,843,989) describes a class of variant forms of theanti-CD40 murine mAb, S2C6, and its use in the treatment of variousdisorders, including cancer and immunological and inflammatory diseases.In addition to enhancing CD40L-mediated stimulation, an anti-CD40antibody described in U.S. Pat. No. 6,838,261 showed enhancement of theinteraction between CD40 and CD40L, and in vivo anti-neoplasticactivity. Although S2C6 by itself will stimulate B cell proliferation ina manner similar to G28-5, S2C6 is distinguished from G28-5 by itsability to increase CD40L binding and the subsequent magnitude of theCD40L-mediated activation signal.

Other murine anti-CD40 mAbs, e.g., described in InternationalPublication Number WO 95/17202, bind CD40 and show efficacy in thetreatment and prevention of disease characterized by neoplastic cellsexpressing CD40. Although murine anti-CD40 antibodies have potentialapplicability as therapeutic agents in the treatment of CD40-relateddiseases in humans, their immunogenicity presents the possibility of aneutralizing antibody response, e.g., a human anti-mouse antibody (HAMA)response which would limit their value.

Thus, there is a need for humanized anti-CD40 antibodies thatspecifically bind defined CD40 epitopes and which show the antigenbinding specificity, affinity, and other desired functionalcharacteristics of the analogous nonhuman anti-CD40 antibody.

BRIEF SUMMARY

The present invention encompasses humanized anti-CD40 antibodies andantigen binding fragments thereof, as well as methods using suchhumanized anti-CD40 antibodies and fragments for the treatment ofdiseases and disorders characterized by cells expressing the CD40surface antigen. Also included are kits and articles of manufacturecomprising a humanized anti-CD40 antibody.

In some embodiments, an isolated antibody or antigen-binding fragmentthat specifically binds to human CD40 is provided. The antibody orantigen-binding fragment includes a heavy chain variable domain and/orlight chain variable region domain. The heavy chain variable regiondomain can include a framework region having an amino acid sequence atleast 90% identical to the amino acid sequence of the human variabledomain heavy chain subgroup III consensus amino acid sequence of SEQ IDNO:2, and at least one CDR having an amino acid sequence at least 90%identical to a corresponding heavy chain CDR of SEQ ID NO:3. The lightchain variable domain can include a framework region having an aminoacid sequence at least 90% identical to the human variable domain lightchain subgroup kappa I consensus amino acid sequence of SEQ ID NO:13,and at least one CDR having an amino acid sequence at least 90%identical to a corresponding light chain CDR of SEQ ID NO:14.

In some embodiments, each heavy chain CDR is at least 90% identical tothe corresponding heavy chain CDR of SEQ ID NO:3. In some embodiments,the heavy chain CDRs include the amino acid sequences of the heavy chainCDR1, CDR2 and CDR3 of SEQ ID NO:3. In some embodiments, each lightchain CDR is at least 90% identical to the corresponding light chain CDRof SEQ ID NO:14. In some embodiments, the light chain CDRs include theamino acid sequences of the CDR1, CDR2 and CDR3 of SEQ ID NO:14.

In some embodiments, the antibody or antigen-binding fragment includes aheavy chain variable domain having the amino acid sequence of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11. In some embodiments, theantibody or antigen-binding fragment includes a light chain variabledomain having the amino acid sequence of SEQ ID NO:14, SEQ ID NO:15, orSEQ ID NO:16. In some embodiments, the antibody or antigen-bindingfragment has the heavy chain variable domain amino acid sequence of SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, and the light chainvariable domain amino acid sequence of SEQ ID NO:14, SEQ ID NO:15, orSEQ ID NO:16.

In some embodiments, the heavy chain variable domain and the light chainvariable domain include the amino acid sequences of SEQ ID NO:3 and SEQID NO:14, respectively; SEQ ID NO:4 and SEQ ID NO:14, respectively; SEQID NO:5 and SEQ ID NO:14, respectively; SEQ ID NO:6 and SEQ ID NO:14,respectively; SEQ ID NO:7 and SEQ ID NO:14, respectively; SEQ ID NO:8and SEQ ID NO:14, respectively; SEQ ID NO:9 and SEQ ID NO:14,respectively; SEQ ID NO:6 and SEQ ID NO:15, respectively; SEQ ID NO:6and SEQ ID NO:16, respectively; SEQ ID NO:7 and SEQ ID NO:16,respectively; SEQ ID NO:10 and SEQ ID NO:14, respectively; SEQ ID NO:11and SEQ ID NO:14, respectively; SEQ ID NO:10 and SEQ ID NO:16,respectively; or SEQ ID NO:11 and SEQ ID NO:16, respectively.

The antibody or antigen-binding fragment can include a human IgGconstant region, such as, for example, an IgG constant region of isotypeIgG1, IgG2, IgG3, or IgG4. The antibody or antigen-binding fragment caninclude a light chain constant domain, such as, for example, a kappaconstant domain.

In some embodiments, the antibody is hu sgn-0, hu sgn-1, hu sgn-2, husgn-4, hu sgn-14, hu sgn-15, hu sgn-16, hu sgn-17, hu sgn-18, hu sgn-19,hu sgn-22, hu sgn-23, hu sgn-26 or hu sgn-27. In some embodiments, theantibody or antigen-binding fragment competes for binding withmonoclonal antibody S2C6 that is secreted by a hybridoma having ATCCAccession No. PTA-110.

The antibody also can be an antigen-binding fragment, such as a Fab, aFab′, a F(ab′)2, a Fv fragment, a diabody, a single-chain antibody, anscFv fragment or an scFv-Fc. The antibody or antigen-binding fragmentcan optionally be labeled or conjugated to a chemotherapeutic agent,such as an auristatin (e.g., MMAE or MMAF).

Also provided is a kit including an anti-CD40 antibody or antigenbinding fragment in a container. The kit can optionally include anadditional component(s), such as instructions for using the antibody todetect CD40 protein in a biological sample.

Pharmaceutical compositions comprising an anti-CD40 antibody orantigen-binding fragment thereof and a pharmaceutically acceptableexcipients(s) are also provided.

In some embodiments, isolated polynucleotides encoding a humanized heavychain variable region and/or a humanized light chain variable region areprovided. A polynucleotide can, for example, encode the heavy chainvariable domain amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,or SEQ ID NO:11. A polynucleotide also can, for example, encode thelight chain variable domain amino acid sequence of SEQ ID NO:14, SEQ IDNO:15, or SEQ ID NO:16.

In some embodiments, isolated polynucleotide encodes the heavy chainvariable domain amino acid sequence and the light chain variable domainamino acid sequence of SEQ ID NO:3 and SEQ ID NO:14, respectively; SEQID NO:4 and SEQ ID NO:14, respectively; SEQ ID NO:5 and SEQ ID NO:14,respectively; SEQ ID NO:6 and SEQ ID NO:14, respectively; SEQ ID NO:7and SEQ ID NO:14, respectively; SEQ ID NO:8 and SEQ ID NO:14,respectively; SEQ ID NO:9 and SEQ ID NO:14, respectively; SEQ ID NO:6and SEQ ID NO:15, respectively; SEQ ID NO:6 and SEQ ID NO:16,respectively; SEQ ID NO:7 and SEQ ID NO:16, respectively; SEQ ID NO:10and SEQ ID NO:14, respectively; SEQ ID NO:11 and SEQ ID NO:14,respectively; SEQ ID NO:10 and SEQ ID NO:16, respectively; or SEQ IDNO:11 and SEQ ID NO:16, respectively.

In some embodiments, methods for inhibiting the growth of cellsexpressing human CD40 antigen are provided. The methods includeadministering an anti-CD40 antibody or an antigen-binding fragment tothe cells, which antibody or antigen-binding fragment binds to the humancell surface CD40 antigen. The binding of the antibody orantigen-binding fragment to the CD40 antigen inhibits the growth ordifferentiation of the cells.

In some embodiments, methods for treating a subject having aCD40-associated disorder are provided. The methods include administeringto the subject an anti-CD40 antibody or an antigen-binding fragment,which antibody or antigen-binding fragment binds to human CD40. Thebinding of the antibody or antigen-binding fragment to CD40 inhibits thegrowth or differentiation of cells of the CD40-associated disorder. TheCD40-associated disorder can be, for example, chronic lymphocyticleukemia, Burkitt's lymphoma, multiple myeloma, a T cell lymphoma,Non-Hodgkin's Lymphoma, Hodgkin's Disease, Waldenstrom'smacroglobulinemia or Kaposi's sarcoma.

In some embodiments, methods for inducing depletion of peripheral Bcells are provided. The methods include administering to the cells ananti-CD40 antibody or an antigen-binding fragment, which antibody orantigen-binding fragment binds to a human cell surface CD40 antigen. Thebinding of the antibody or antigen-binding fragment to the CD40 antigeninduces depletion of the cells. The peripheral B cells can, for example,exhibit autoimmune reactivity in a subject.

The invention will best be understood by reference to the followingdetailed description including the preferred embodiments, taken inconjunction with the accompanying drawings and sequence listing. Thediscussion below is descriptive, illustrative and exemplary and is notto be taken as limiting the scope defined by any of the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the polypeptide (SEQ ID NO:18) and the coding (SEQID NO:17) and complementary DNA sequences of the heavy chain of ahumanized anti-CD40 antibody. The polypeptide sequence is annotated toindicate the position of the leader sequence, the variable region, andthe human IgG₁ constant region. FIG. 1C shows the polypeptide (SEQ IDNO:21) and the coding (SEQ ID NO:20) and complementary DNA sequences ofthe light chain of a humanized anti-CD40 antibody. The polypeptidesequence is annotated to indicate the position of the leader sequence,the variable region, and the human kappa constant region.

FIG. 2 shows the effect of treatment with a control antibody, a murineanti-CD40 antibody, and a humanized anti-CD40 antibody on tumor volumemeasured over a two-week period, with treatment beginning 13 dayspost-tumor tumor transplant.

FIG. 3 shows the effect of treatment with a control antibody, a murineanti-CD40 antibody, and a humanized anti-CD40 antibody, on survival oftumor-bearing mice.

DETAILED DESCRIPTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections whichfollow.

When trade names are used herein, the trade name also refers to thetrade name product formulation, the generic drug, and the activepharmaceutical ingredient(s) of the trade name product, unless otherwiseindicated by context.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art pertinent to the methods and compositions described.

DEFINITIONS

The terms “CD40” and “CD40 surface antigen” refer to a 50 kDglycoprotein expressed on the surface of normal and neoplastic B cells,which acts as a receptor for signals involved in cellular proliferationand differentiation and is sometimes referred to as Bp50 (Ledbetter etal., 1987, J. Immunol. 138:788-785). A cDNA molecule encoding CD40 hasbeen isolated from a library prepared from the Burkitt lymphoma cellline Raji (Stamenkovic et al., 1989, EMBO J. 8:1403). A cell thatexpresses CD40 is any cell characterized by the surface expression ofCD40, including, but not limited to, normal and neoplastic B cells,interdigitating cells, basal epithelial cells, carcinoma cells,macrophages, endothelial cells, follicular dendritic cells, tonsilcells, and bone marrow-derived plasma cells. In some embodiments, theCD40 molecule is a human CD40 molecule.

The terms, “CD40 antigen epitope” and“CD40 epitope”, as used herein,refer to a molecule (e.g., a peptide) or a fragment of a moleculecapable of immunoreactivity with an anti-CD40 antibody and, for example,includes a CD40 antigenic determinant recognized by the S2C6 monoclonalantibody. CD40 antigen epitopes can be included in proteins, proteinfragments, peptides or the like. The epitopes are most commonlyproteins, short oligopeptides, oligopeptide mimics (i.e., organiccompounds that mimic antibody binding properties of the CD40 antigen),or combinations thereof.

As used herein, “specific binding” and “specifically binds” refer toantibody binding to a predetermined antigen. Typically, the antibodybinds with an affinity of at least about 1×10⁷ M⁻¹, and binds to thepredetermined antigen with an affinity that is at least two-fold greaterthan its affinity for binding to a non-specific antigen (e.g., BSA,casein) other than the predetermined antigen or a closely-relatedantigen.

“Native antibodies” and “native immunoglobulins” are defined herein asheterotetrameric glycoproteins, typically of about 150,000 daltons,composed of two identical light (L) chain and two identical heavy (H)chains. Each light chain is covalently linked to a heavy chain by onedisulfide bond to form a heterodimer. The heterotetramer is formed bycovalent disulfide linkage between the two identical heavy chains ofsuch heterodimers. Although the light and heavy chains are linkedtogether by one disulfide bond, the number of disulfide linkages betweenthe two heavy chains varies by immunoglobulin isotype. Each heavy andlight chain also has regularly spaced intrachain disulfide bridges. Eachheavy chain has at the amino-terminus a variable domain (V_(H)),followed by three or four constant domains (C_(H)1, C_(H)2, C_(H)3, andC_(H)4), as well as a hinge region between C_(H)1 and C_(H)2. Each lightchain has two domains, an amino-terminal variable domain (V_(L)) and acarboxy-terminal constant domain (C_(L)). The V_(L) domain associatesnon-covalently with the V_(H) domain, whereas the C_(L) domain iscommonly covalently linked to the C_(H)1 domain via a disulfide bond.Particular amino acid residues are believed to form an interface betweenthe light and heavy chain variable domains (Chothia et al., 1985, J.Mol. Biol. 186:651-663.)

The term “hypervariable” refers to the fact that certain sequenceswithin the variable domains differ extensively in sequence amongantibodies and contain residues that are directly involved in thebinding and specificity of each particular antibody for its specificantigenic determinant. Hypervariability, both in the light chain and theheavy chain variable domains, is concentrated in three segments known ascomplementarity determining regions (CDRs) or hypervariable loops(HVLs). CDRs are defined by sequence comparison in Kabat et al., 1991,In: Sequences of Proteins of Immunological Interest, 5^(th) Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., whereasHVLs are structurally defined according to the three-dimensionalstructure of the variable domain, as described by Chothia and Lesk,1987, J. Mol. Biol. 196: 901-917. Where these two methods result inslightly different identifications of a CDR, the structural definitionis preferred. As defined by Kabat, CDR-L1 is positioned at aboutresidues 24-34, CDR-L2, at about residues 50-56, and CDR-L3, at aboutresidues 89-97 in the light chain variable domain; CDR-H1 is positionedat about residues 31-35, CDR-H2 at about residues 50-65, and CDR-H3 atabout residues 95-102 in the heavy chain variable domain.

The three CDRs within each of the heavy and light chains are separatedby framework regions (FR), which contain sequences that tend to be lessvariable. From the amino terminus to the carboxy terminus of the heavyand light chain variable domains, the FRs and CDRs are arranged in theorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The largely β-sheetconfiguration of the FRs brings the CDRs within each of the chains toclose proximity to each other as well as to the CDRs from the otherchain. The resulting conformation contributes to the antigen bindingsite (see Kabat et al., 1991, NIH Publ. No. 91-3242, Vol. I, pages647-669), although not all CDR residues are necessarily directlyinvolved in antigen binding.

FR residues and Ig constant domains are not directly involved in antigenbinding, but contribute to antigen binding and/or mediate antibodyeffector function. Some FR residues can have a significant effect onantigen binding in at least three ways: by noncovalently bindingdirectly to an epitope, by interacting with one or more CDR residues,and by affecting the interface between the heavy and light chains. Theconstant domains are not directly involved in antigen binding butmediate various Ig effector functions, such as participation of theantibody in antibody dependent cellular cytotoxicity (ADCC), complementdependent cytotoxicity (CDC) and antibody dependent cellularphagocytosis (ADCP).

The light chains of vertebrate immunoglobulins are assigned to one oftwo clearly distinct classes, kappa (κ) and lambda (λ), based on theamino acid sequence of the constant domain. By comparison, the heavychains of mammalian immunoglobulins are assigned to one of five majorclasses, according to the sequence of the constant domains: IgA, IgD,IgE, IgG, and IgM. IgG and IgA are further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavychain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of the classes of nativeimmunoglobulins are well known.

The terms, “antibody”, “anti-CD40 antibody”, “humanized anti-CD40antibody”, and “variant humanized anti-CD40 antibody” are used herein inthe broadest sense and specifically encompass monoclonal antibodies(including full length monoclonal antibodies), polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies), and antibodyfragments such as variable domains and other portions of antibodies thatexhibit a desired biological activity, e.g., CD40 binding.

The term “monoclonal antibody” (mAb) refers to an antibody obtained froma population of substantially homogeneous antibodies; that is, theindividual antibodies comprising the population are identical except fornaturally occurring mutations that may be present in minor amounts.Monoclonal antibodies are highly specific, being directed against asingle antigenic determinant, also referred to as an epitope. Themodifier “monoclonal” is indicative of a substantially homogeneouspopulation of antibodies directed to the identical epitope and is not tobe construed as requiring production of the antibody by any particularmethod. Monoclonal antibodies can be made by any technique ormethodology known in the art; for example, the hybridoma method firstdescribed by Köhler et al., 1975, Nature 256:495, or recombinant DNAmethods known in the art (see, e.g., U.S. Pat. No. 4,816,567). Inanother example, monoclonal antibodies can also be isolated from phageantibody libraries, using techniques described in Clackson et al., 1991,Nature 352: 624-628, and Marks et al., 1991, J. Mol. Biol. 222: 581-597.

In contrast, the antibodies in a preparation of polyclonal antibodiesare typically a heterogeneous population of immunoglobulin isotypesand/or classes and also exhibit a variety of epitope specificity.

The term “chimeric” antibody as used herein is a type of monoclonalantibody in which a portion of or the complete amino acid sequence inone or more regions or domains of the heavy and/or light chain isidentical with, homologous to, or a variant of the correspondingsequence in a monoclonal antibody from another species or belonging toanother immunoglobulin class or isotype, or from a consensus sequence.Chimeric antibodies include fragments of such antibodies, provided thatthe antibody fragment exhibits the desired biological activity of itsparent antibody, for example binding to the same epitope (see, e.g.,U.S. Pat. No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad.Sci. USA 81: 6851-6855).

The terms, “antibody fragment”, “anti-CD40 antibody fragment”,“humanized anti-CD40 antibody fragment”, “variant humanized anti-CD40antibody fragment” refer to a portion of a full length anti-CD40antibody, in which a variable region or a functional capability isretained, for example, specific CD40 epitope binding. Examples ofantibody fragments include, but are not limited to, a Fab, Fab′,F(ab′)₂, Fd, Fv, scFv and scFv-Fc fragment, a diabody, a linearantibody, a single-chain antibody, a minibody, a diabody formed fromantibody fragments, and multispecific antibodies formed from antibodyfragments.

Certain types of antibody fragments can be generated by enzymatictreatment of a full-length antibody. Papain digestion of antibodiesproduces two identical antigen-binding fragments called “Fab” fragments,each with a single antigen-binding site, and a residual “Fc” fragment,so called because of its ability to crystallize readily. The Fabfragment also contains the constant domain of the light chain and theC_(H)1 domain of the heavy chain. Pepsin treatment yields a F(ab′)₂fragment that has two antigen-binding sites and is still capable ofcross-linking antigen.

Fab′ fragments differ from Fab fragments by the presence of a fewadditional residues at the C-terminus of the C_(H)1 domain, includingone or more cysteines from the antibody hinge region. Fab-SH is thedesignation herein for a Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragments arepairs of Fab′ fragments linked by cysteine residues in the hinge region.Other chemical couplings of antibody fragments are also known.

“Fv” is a minimum antibody fragment that contains a completeantigen-recognition and binding site consisting of a dimer of one heavyand one light chain variable domain in tight, non-covalent association.In this configuration, the three CDRs of each variable domain interactto define an antigen-biding site on the surface of the V_(H)-V_(L)dimer. Collectively, the six CDRs confer antigen-binding specificity tothe antibody.

A “single-chain Fv” or “scFv” antibody fragment is a single chain Fvvariant comprising the V_(H) and V_(L) domains of an antibody, in whichthe domains are present in a single polypeptide chain and which iscapable of recognizing and binding antigen. The scFv polypeptideoptionally contains a polypeptide linker positioned between the V_(H)and V_(L) domains that enables the scFv to form a desiredthree-dimensional structure for antigen binding (see, e.g., Pluckthun,1994, In The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburgand Moore eds., Springer-Verlag, New York, pp. 269-315).

The term “diabodies” refers to small antibody fragments having twoantigen-binding sites. Each fragment contains a heavy chain variabledomain (V_(H)) concatenated to a light chain variable domain (V_(L)). Byusing a linker that is too short to allow pairing between the twodomains on the same chain, the linked V_(H)-V_(L) domains are forced topair with complementary domains of another chain, creating twoantigen-binding sites. Diabodies are described more fully, for example,in EP 404,097; WO 93/11161; and Hollinger et al., 1993, Proc. Natl.Acad. Sci. USA 90: 6444-6448.

The term “linear antibodies” refers to antibodies that comprise a pairof tandem Fd segments (V_(H)-C_(H)1-V_(H)-C_(H)1) that form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific as described in, for example, Zapata et al. 1995, ProteinEng. 8(10):1057-1062.

A humanized antibody or a humanized antibody fragment includes animmunoglobulin amino acid sequence variant, or fragment thereof, whichis capable of binding to a predetermined antigen and which, comprisesone or more FRs having substantially the amino acid sequence of a humanimmunoglobulin and one or more CDRs having substantially the amino acidsequence of a non-human immunoglobulin. This non-human amino acidsequence is referred to herein as an “import” sequence, which istypically taken from an “import” antibody domain, particularly avariable domain. In general, a humanized antibody includes at least theCDRs or HVLs of a non-human antibody, inserted between the FRs of ahuman heavy or light chain variable domain. In certain aspects, ahumanized anti-CD40 antibody contains CDR and/or HVL residues orsequences derived from the murine monoclonal antibody S2C6 insertedbetween the FRs of human consensus sequence heavy and light chainvariable domains.

In another aspect, a humanized anti-CD40 antibody comprisessubstantially all of at least one, and typically two, variable domains(such as contained, for example, in Fab, Fab′, F(ab′)₂, Fabc, and Fvfragments) in which all, or substantially all, of the CDRs correspond tothose of a non-human immunoglobulin and all, or substantially all, ofthe FRs are those of a human immunoglobulin consensus sequence. Inanother aspect, a humanized anti-CD40 antibody also includes at least aportion of an immunoglobulin Fc region, typically that of a humanimmunoglobulin. Ordinarily, the antibody will contain both the lightchain as well as at least the variable domain of a heavy chain. Theantibody also may include one or more of the C_(H)1, hinge, C_(H)2,C_(H)3, and/or C_(H)4 regions of the heavy chain, as appropriate.

A humanized anti-CD40 antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂. For example, theconstant domain can be a complement fixing constant domain where it isdesired that the humanized antibody exhibit cytotoxic activity, and theisotype is typically IgG₁. Where such cytotoxic activity is notdesirable, the constant domain may be of another isotype, e.g., IgG₂. Analternative humanized anti-CD40 antibody can comprise sequences frommore than one immunoglobulin class or isotype, and selecting particularconstant domains to optimize desired effector functions is within theordinary skill in the art.

The FRs and CDRs, or HVLs, of a humanized anti-CD40 antibody need notcorrespond precisely to the parental sequences. For example, one or moreresidues in the import CDR, or HVL, or the consensus FR sequence may bealtered (e.g., mutagenized) by substitution, insertion or deletion suchthat the resulting amino acid residue is no longer identical to theoriginal residue in the corresponding position in either parentalsequence. Such alterations, however, typically will not be extensive.Usually, at least 75% of the humanized antibody residues will correspondto those of the parental consensus FR and import CDR sequences, moreoften at least 90%, and most frequently greater than 95%, or greaterthan 98% or greater than 99%.

Immunoglobulin residues that affect the interface between heavy andlight chain variable regions (“the V_(L)-V_(H) interface”) are thosethat affect the proximity or orientation of the two chains with respectto one another. Certain residues that may be involved in interchaininteractions include V_(L) residues 34, 36, 38, 44, 46, 87, 89, 91, 96,and 98 and V_(H) residues 35, 37, 39, 45, 47, 91, 93, 95, 100, and 103(utilizing the numbering system set forth in Kabat et al., Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md., 1987)). Additional residues include V_(L) residues 43 and85, and V_(H) residues 43 and 60, as disclosed in U.S. Pat. No.6,407,213, which is hereby incorporated by reference in its entirety.While these residues are indicated for human IgG only, they areapplicable across species. Import antibody residues that are reasonablyexpected to be involved in interchain interactions are selected forsubstitution into the consensus sequence.

The terms “consensus sequence” and “consensus antibody” as used hereinrefer to an amino acid sequence which comprises the most frequentlyoccurring amino acid residue at each location in all immunoglobulins ofany particular class, isotype, or subunit structure, e.g., a humanimmunoglobulin variable domain. The consensus sequence may be based onimmunoglobulins of a particular species or of many species. A“consensus” sequence, structure, or antibody is understood to encompassa consensus human sequence as described in certain embodiments, and torefer to an amino acid sequence which comprises the most frequentlyoccurring amino acid residues at each location in all humanimmunoglobulins of any particular class, isotype, or subunit structure.Provided are consensus human structures and consensus structures whichconsider other species in addition to human. Thus, the consensussequence contains an amino acid sequence having at each position anamino acid that is present in one or more known immunoglobulins, butwhich may not exactly duplicate the entire amino acid sequence of anysingle immunoglobulin. The variable region consensus sequence is notobtained from any naturally produced antibody or immunoglobulin. Usefulconsensus sequences include a human variable light chain kappa Iconsensus sequence (SEQ ID NO:13) and a human variable heavy chainsubgroup III consensus sequence (SEQ ID NO:2), derived from the dataprovided in Kabat et al., 1991, Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md., and variants thereof. The FRs of heavy and light chainconsensus sequences, and variants thereof, provide useful sequences forthe preparation of humanized anti-CD40 antibodies. See, for example,U.S. Pat. Nos. 6,037,454 and 6,054,297. In certain embodiments, the FRused to prepare the humanized antibodies were derived from consensussequences for a human variable light chain kappa I consensus sequenceand for a human variable heavy chain subgroup III consensus sequence.

As used herein, “variant”, “anti-CD40 variant”, “humanized anti-CD40variant”, or “variant humanized anti-CD40” each refers to a humanizedanti-CD40 antibody having at least a heavy chain variable CDR or HVLsequence derived from the murine monoclonal antibody S2C6 and FRsequences derived from human consensus sequences. Variants include thosehaving one or more amino acid changes in one or both light chain orheavy chain variable domains, provided that the amino acid change doesnot substantially impair binding of the antibody to CD40. Humanizedanti-CD40 variants typically include amino acid substitutions thatimprove antibody performance by allowing improved folding of theantibody molecule.

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of the antibody's natural environment are thosematerials that may interfere with diagnostic or therapeutic uses of theantibody, and can be enzymes, hormones, or other proteinaceous ornonproteinaceous solutes. In one aspect, the antibody will be purified:

-   -   (a) to greater than 95% isolation by weight of antibody as        determined by the Lowry method, and in another aspect, more than        99% isolation by weight, or    -   (b) to a degree of isolation sufficient to obtain at least 15        residues of N-terminal or internal amino acid sequence by use of        a spinning cup sequenator, or    -   (c) to homogeneity by SDS-PAGE under reducing or nonreducing        conditions as visualized using Coomassie blue or, preferably,        silver stain.

An isolated antibody includes an antibody in situ within recombinantcells, since at least one component of the antibody's naturalenvironment will not be present. Ordinarily however, an isolatedantibody will be prepared by at least one purification step.

The term “antibody performance” refers to factors that contribute toantibody recognition of antigen or the effectiveness of an antibody invivo. Changes in the amino acid sequence of an antibody can affectantibody properties such as folding, and can influence physical factorssuch as initial rate of antibody binding to antigen (v_(o)),dissociation constant of the antibody from antigen (Kd), affinityconstant of the antibody for the antigen, conformation of the antibody,protein stability, and half life of the antibody.

The term “epitope tagged” when used herein, refers to an anti-CD40antibody fused to an “epitope tag”. An “epitope tag” is a polypeptidehaving a sufficient number of amino acids to provide an epitope forantibody production, yet is designed such that it does not interferewith the desired activity of the humanized anti-CD40 antibody. Theepitope tag is usually sufficiently unique such that an antibody raisedagainst the epitope tag does not substantially cross-react with otherepitopes. Suitable tag polypeptides generally contain at least 6 aminoacid residues and usually contain about 8 to 50 amino acid residues, orabout 9 to 30 residues. Examples of epitope tags and the antibody thatbinds the epitope include the flu HA tag polypeptide and its antibody12CA5 (Field et al., 1988 Mol. Cell. Biol. 8: 2159-2165; c-myc tag and8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., 1985,Mol. Cell. Biol. 5(12):3610-3616; and Herpes simplex virus glycoproteinD (gD) tag and its antibody (Paborsky et al. 1990, Protein Engineering3(6): 547-553). In certain embodiments, the epitope tag is a “salvagereceptor binding epitope”. As used herein, the term “salvage receptorbinding epitope” refers to an epitope of the Fc region of an IgGmolecule (such as IgG₁, IgG₂, IgG₃, or IgG₄) that is responsible forincreasing the in vivo serum half-life of the IgG molecule.

The term “cytotoxic agent” refers to a substance that inhibits orprevents the function of cells and/or causes destruction of cells. Theterm is intended to include radioactive isotopes (such as I¹³¹, I¹²⁵,Y⁹⁰, and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant, or animalorigin, and fragments thereof. Such cytotoxic agents can be coupled toan antibody, e.g., a humanized anti-CD40 antibody, using known, standardprocedures, and used, for example, to treat a patient indicated fortherapy with the antibody.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such a thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan, and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphoramide, and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin, and bizelesin syntheticanalogues); cryptophycines (particularly cryptophycin 1 and cryptophycin8); dolastatin, auristatins, (including analogues monomethyl-auristatinE and monomethyl-auristatin F); duocarmycin (including the syntheticanalogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin;sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil,chlornaphazine, cholophosphamide, estramustine, ifosfamide,mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,novembichin, phenesterine, prednimustine; trofosfamide, uracil mustard;nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, ranimustine; antibiotics such as the enediyne antibiotics(e.g., calicheamicin, especially calichemicin gamma1I and calicheamicinphiI1, see for example, Agnew, Chem. Intl. Ed. Engl., 33:183-186;dynemicin, including dynemicin A; bisphosphonates, such as clodronate;esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromomophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(Adriamycin™) (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, anddeoxydoxorubicin), epirubucin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycine, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such a methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adranals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; democolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids suchas maytansine and ansamitocins; mitoguazone, mitoxantrone; mopidamol;nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitabronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.,paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine (Gemzar™); 6-thioguanine; mercaptopurine;methotrexate; platinum analogs such as cisplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine (Navelbine™); novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoids such as retinoic acid; capecitabine; and pharmaceuticallyacceptable salts, acids, or derivatives of any of the above. Alsoincluded in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including Nolvadex™), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (Fareston™); aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrolacetate (Megace™), exemestane, formestane, fadrozole, vorozole(Rivisor™), letrozole (Femara™), and anastrozole (Arimidex™); andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; and pharmaceutically acceptable salts, acids, orderivatives of any of the above.

The term “prodrug” as used herein refers to a precursor or derivativeform of a pharmaceutically active substance that is less cytotoxic totumor cells compared to the parent drug and is capable of beingenzymatically activated or converted into the more active form. See, forexample, Wilman, 1986, “Prodrugs in Cancer Chemotherapy”, In BiochemicalSociety Transactions, 14, pp. 375-382, 615th Meeting Belfast and Stellaet al., 1985, “Prodrugs: A Chemical Approach to Targeted Drug Delivery,In: “Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267,Humana Press. Useful prodrugs include, but are not limited to,phosphate-containing prodrugs, thiophosphate-containing prodrugs,sulfate-containing prodrugs peptide-containing prodrugs, D-aminoacid-modified prodrugs, glycosylated prodrugs, β-lactam-containingprodrugs, optionally substituted phenoxyacetamide-containing prodrugs,and optionally substituted phenylacetamide-containing prodrugs,5-fluorocytosine and other 5-fluorouridine prodrugs that can beconverted into the more active cytotoxic free drug. Examples ofcytotoxic drugs that can be derivatized into a prodrug form include, butare not limited to, those chemotherapeutic agents described above.

The term “label” refers to a detectable compound or composition that isconjugated directly or indirectly to the antibody. The label may itselfbe detectable (e.g., radioisotope labels or fluorescent labels) or, inthe case of an enzymatic label, may catalyze chemical alteration of asubstrate compound or composition that is detectable. Labeled humanizedanti-CD40 antibody can be prepared and used in various applicationsincluding in vitro and in vivo diagnostics.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids, and/or surfactant. Liposomes are useful for delivery to amammal of a compound or formulation, such as a humanized anti-CD40antibody disclosed herein, optionally, coupled to or in combination withone or more pharmaceutically active agents. The components of theliposome are commonly arranged in a bilayer formation, similar to thelipid arrangement of biological membranes.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe antibody nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the antibody where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “control sequences” refers to polynucleotide sequencesnecessary for expression of an operably linked coding sequence in aparticular host organism. The control sequences suitable for use inprokaryotic cells include, for example, promoter, operator, and ribosomebinding site sequences. Eukaryotic control sequences include, but arenot limited to, promoters, polyadenylation signals, and enhancers. Thesecontrol sequences can be utilized for expression and production ofhumanized anti-CD40 antibody in prokaryotic and eukaryotic host cells.

A nucleic acid sequence is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,a nucleic acid presequence or secretory leader is operably linked to anucleic acid encoding a polypeptide if it is expressed as a preproteinthat participates in the secretion of the polypeptide; a promoter orenhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, “operably linked” means that the DNA sequencesbeing linked are contiguous, and, in the case of a secretory leader,contiguous and in reading frame. However, enhancers are optionallycontiguous. Linking can be accomplished by ligation at convenientrestriction sites. If such sites do not exist, synthetic oligonucleotideadaptors or linkers can be used.

As used herein, the expressions “cell”, “cell line”, and “cell culture”are used interchangeably and all such designations include the progenythereof. Thus, “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or naturallyoccurring mutations. Mutant progeny that have the same function orbiological activity as screened for in the originally transformed cellare included. Where distinct designations are intended, it will be clearfrom the context.

The term “mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domesticated and farm animals,and zoo, sports, or pet animals, such as dogs, horses, cats, cows, andthe like. Preferably, the mammal is human.

A “disorder”, as used herein, is any condition that would benefit fromtreatment with a humanized anti-CD40 antibody described herein. Thisincludes chronic and acute disorders or diseases including thosepathological conditions that predispose the mammal to the disorder inquestion. Non-limiting examples or disorders to be treated hereininclude cancer, hematological malignancies, benign and malignant tumors,leukemias and lymphoid malignancies and inflammatory, angiogenic andimmunologic disorders.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.

As used herein, the term “CD40-associated disorder” or “CD40-associateddisease” refers to a condition in which modification or elimination ofcells expressing CD40 is indicated. These include CD40-expressing cellsdemonstrating abnormal proliferation or CD40-expressing cells that areassociated with cancerous or malignant growth. More particular examplesof cancers that demonstrate abnormal expression of CD40 antigen includeB lymphoblastoid cells, Burkitt's lymphoma, multiple myeloma, T celllymphomas, Kaposi's sarcoma, osteosarcoma, epidermal and endothelialtumors, pancreatic, lung, breast, ovarian, colon, prostate, head andneck, skin (melanoma), bladder, and kidney cancers. Such disordersinclude, but are not limited to, leukemias, lymphomas, including B celllymphoma and non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom'smacroglobulinemia; solid tumors, including sarcomas, such asosteosarcoma, Ewing's sarcoma, malignant melanoma, adenocarcinoma,including ovarian adenocarcinoma, Kaposi's sarcoma/Kaposi's tumor andsquamous cell carcinoma.

A CD40-associated disorder also includes diseases and disorders of theimmune system, such as auto-immune disorders and inflammatory disorders.Such conditions include, but are not limited to, rheumatoid arthritis(RA), systemic lupus erythematosus (SLE), scleroderma, Sjogren'ssyndrome, multiple sclerosis, inflammatory bowel disease (e.g.,ulcerative colitis and Crohn's disease), pulmonary inflammation, asthma,and idiopathic thrombocytopenic purara (ITP).

The phrase “arrests the growth of” or “growth inhibitory” when usedherein refers to inhibiting growth or proliferation of a cell,especially a neoplastic cell type expressing the CD40 antigen. Thus,growth inhibition, for example, significantly reduces the percentage ofneoplastic cells in S phase.

The term “intravenous infusion” refers to introduction of an agent intothe vein of an animal or human patient over a period of time greaterthan approximately 15 minutes, generally between approximately 30 to 90minutes.

The term “intravenous bolus” or “intravenous push” refers to drugadministration into a vein of an animal or human such that the bodyreceives the drug in approximately 15 minutes or less, generally 5minutes or less.

The term “subcutaneous administration” refers to introduction of anagent under the skin of an animal or human patient, preferable within apocket between the skin and underlying tissue, by relatively slow,sustained delivery from a drug receptacle. Pinching or drawing the skinup and away from underlying tissue may create the pocket.

The term “subcutaneous infusion” refers to introduction of a drug underthe skin of an animal or human patient, preferably within a pocketbetween the skin and underlying tissue, by relatively slow, sustaineddelivery from a drug receptacle for a period of time including, but notlimited to, 30 minutes or less, or 90 minutes or less. Optionally, theinfusion may be made by subcutaneous implantation of a drug deliverypump implanted under the skin of the animal or human patient, whereinthe pump delivers a predetermined amount of drug for a predeterminedperiod of time, such as 30 minutes, 90 minutes, or a time periodspanning the length of the treatment regimen.

The term “subcutaneous bolus” refers to drug administration beneath theskin of an animal or human patient, where bolus drug delivery is lessthan approximately 15 minutes; in another aspect, less than 5 minutes,and in still another aspect, less than 60 seconds. In yet even anotheraspect, administration is within a pocket between the skin andunderlying tissue, where the pocket may be created by pinching ordrawing the skin up and away from underlying tissue.

The term “therapeutically effective amount” is used to refer to anamount of an active agent having beneficial patient outcome, forexample, a growth arrest effect or causes the deletion of the cell. Inone aspect, the therapeutically effective amount has apoptotic activity,or is capable of inducing cell death. In another aspect, thetherapeutically effective amount refers to a target serum concentrationthat has been shown to be effective in, for example, slowing diseaseprogression. Efficacy can be measured in conventional ways, depending onthe condition to be treated. For example, in neoplastic diseases ordisorders characterized by cells expressing CD40, efficacy can bemeasured by assessing the time to disease progression (TTP), ordetermining the response rates (RR).

The terms “treatment” and “therapy” and the like, as used herein, aremeant to include therapeutic as well as prophylactic, or suppressivemeasures for a disease or disorder leading to any clinically desirableor beneficial effect, including but not limited to alleviation or reliefof one or more symptoms, regression, slowing or cessation of progressionof the disease or disorder. Thus, for example, the term treatmentincludes the administration of an agent prior to or following the onsetof a symptom of a disease or disorder thereby preventing or removing oneor more signs of the disease or disorder. As another example, the termincludes the administration of an agent after clinical manifestation ofthe disease to combat the symptoms of the disease. Further,administration of an agent after onset and after clinical symptoms havedeveloped where administration affects clinical parameters of thedisease or disorder, such as the degree of tissue injury or the amountor extent of metastasis, whether or not the treatment leads toamelioration of the disease, comprises “treatment” or “therapy” as usedherein.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

The abbreviation “AFP” refers todimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylenediamine.

The abbreviation “MMAE” refers to monomethyl auristatin E.

The abbreviation “AEB” refers to an ester produced by reactingauristatin E with paraacetyl benzoic acid.

The abbreviation “AEVB” refers to an ester produced by reactingauristatin E with benzoylvaleric acid.

The abbreviation “MMAF” refers todovaline-valine-dolaisoleunine-dolaproine-phenylalanine.

Antibodies

Described and disclosed herein are humanized anti-CD40 antibodies, andcompositions and articles of manufacture comprising a humanizedanti-CD40 antibody. Also described are binding agents that include anantigen-binding fragment of a humanized anti-CD40 antibody. Thehumanized anti-CD40 antibodies and binding agents can arrest the growthof cells, cause the deletion of cells expressing CD40 or otherwiseinduce or cause a cytotoxic or cytostatic effect on target cells. Thehumanized anti-CD40 antibodies and binding agents can be used in thetreatment of a variety of diseases or disorders characterized by theproliferation of cells expressing the CD40 surface antigen.

A humanized anti-CD40 antibody and a CD40 binding agent each includes atleast a portion that specifically recognizes a CD40 epitope (i.e., anantigen-binding fragment). In some embodiments a humanized anti-CD40antibody or a CD40 binding agent includes an antigen-binding fragmentthat competes for binding with antibody S2C6.

In some embodiments, the antigen-binding fragment can, for example,block proliferation or otherwise arrest the growth of a cell or causeits depletion, death, or otherwise its deletion, for example, throughbinding the CD40 surface antigen. For example, in T and B cellmalignancies, anti-tumor effects (e.g., growth arrest with or withoutcell deletion or apoptosis) often result when malignant cells areexposed to stimuli that lead to activation of normal lymphocytes. Thisactivation-induced growth arrest has been observed with signals througheither antigen receptors or costimulatory receptors (see, e.g., Ashwellet al., 1987, Science 237:61; Bridges et al., 1987, J. Immunol.139:4242; Page and Defranco, 1988, J. Immunol. 140:3717; and Beckwith etal., 1990, J. Natl. Cancer Inst. 82:501). CD40 stimulation, as a resultof specific binding by either antibody or soluble ligand, inhibits Bcell lymphoma growth (see, e.g., Funakoshi et al., 1994, Blood83:2787-2794). Agents that inhibit malignant cell growth in this way andthat are directed against the CD40 surface antigen are examples ofappropriate agents.

CD40 specific agents include an antigen-binding fragment of a humanizedanti-CD40 antibody that binds to CD40 (e.g., human CD40 or a variantthereof). The CD40 specific agents and antibodies can be optionallyconjugated with or fused to a cytotoxic or chemotherapeutic agent. Inaspects where the humanized antibody binds to the CD40 surface antigenand causes depletion of the CD40 expressing cell types, binding isgenerally characterized by homing to the CD40 surface antigen cell invivo. Suitable binding agents bind the CD40 antigen with sufficientaffinity and/or avidity such that the CD40 specific agent is useful as atherapeutic agent by specifically targeting a cell expressing theantigen.

In one aspect, the agent is a humanized antibody containing the CDRs ofthe murine monoclonal antibody S2C6. (The S2C6 antibody is described,for example, by Paulie et al., 1984, Cancer Immunol. Immunother.17:165-179.) The S2C6 antibody has been shown to exert an agonistactivity on human peripheral B cells as demonstrated by the antibody'sability to stimulate primary B cell proliferation in a dose dependentmanner (see, e.g., Paulie et al., 1989, J. Immunol. 142:590-595), aswell as anti-neoplastic activity in vivo (see, e.g., U.S. Pat. No.6,838,261).

In some aspects, the humanized antibody increases the binding of CD40ligand to CD40 by at least 45%, by at least 50%, by at least 60% or byat least 75%. A method of determining increases in binding of CD40ligand to CD40 are disclosed in U.S. Pat. No. 6,838,261 (the disclosureof which is incorporated by reference herein).

In some embodiments, the humanized anti-CD40 antibodies, includingantigen-binding fragments thereof, such as heavy and light chainvariable domains, comprise an amino acid sequence of the residuesderived from the CDRs or HVLs of the S2C6 murine antibody (see, e.g.,U.S. Pat. No. 6,838,261) and amino acid residues derived from frameworkregions of a human immunoglobulin. In one aspect, the human frameworkregion amino acids are derived from human consensus sequences for theheavy chain subgroup III variable domain and the kappa light chainvariable as described in U.S. Pat. No. 6,037,454. The humanizedanti-CD40 antibodies optionally include specific amino acidsubstitutions in the consensus framework regions.

The specific substitution of amino acid residues in these frameworkpositions can improve various aspects of antibody performance includingbinding affinity and/or stability, over that demonstrated in humanizedantibodies formed by “direct swap” of CDRs or HVLs into the humanconsensus framework regions, as shown in the examples below.

In some embodiments, the humanized anti-CD40 antibodies disclosed hereincomprise at least a heavy or light chain variable domain comprising theCDRs or HVLs of the murine monoclonal antibody S2C6 and the FRs of thehuman consensus heavy and light chain variable domains having thespecific substitutions described in Table 5. An alignment of thevariable heavy chain amino acid sequences having substitutions andvariable light chain amino acid sequences having substitutions are shownin Tables 3 and 4, respectively. These sequences include a heavy chainvariable domain having the amino acid sequence of SEQ ID NO:3 and alight chain variable domain having the amino acid sequence of SEQ IDNO:14.

In certain embodiments, the humanized anti-CD40 antibody is an antibodyfragment. Various techniques have been developed for the production ofantibody fragments. Fragments can be derived via proteolytic digestionof intact antibodies (see, e.g., Morimoto et al., 1992, Journal ofBiochemical and Biophysical Methods 24:107-117; and Brennan et al.,1985, Science 229:81). Alternatively, the fragments can be produceddirectly in recombinant host cells. For example, Fab′-SH fragments canbe directly recovered from E. coli and chemically coupled to formF(ab′)₂ fragments (see, e.g., Carter et al., 1992, Bio/Technology10:163-167). By another approach, F(ab′)₂ fragments can be isolateddirectly from recombinant host cell culture. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner.

Certain embodiments include an F(ab′)₂ fragment of a humanized anti-CD40antibody comprising a heavy chain variable domain amino acid sequenceand a light chain variable domain amino acid sequence of SEQ ID NO:3 andSEQ ID NO:14, respectively; SEQ ID NO:4 and SEQ ID NO:14, respectively;SEQ ID NO:5 and SEQ ID NO:14, respectively; SEQ ID NO:6 and SEQ IDNO:14, respectively; SEQ ID NO:7 and SEQ ID NO:14, respectively; SEQ IDNO:8 and SEQ ID NO:14, respectively; SEQ ID NO:9 and SEQ ID NO:14,respectively; SEQ ID NO:6 and SEQ ID NO:15, respectively; SEQ ID NO:6and SEQ ID NO:16, respectively; SEQ ID NO:7 and SEQ ID NO:16,respectively; SEQ ID NO:10 and SEQ ID NO:14, respectively; SEQ ID NO:11and SEQ ID NO:14, respectively; SEQ ID NO:10 and SEQ ID NO:16,respectively; or SEQ ID NO:11 and SEQ ID NO:16, respectively. Suchembodiments can include an intact antibody comprising such an F(ab′)₂.

Other embodiments include a F(ab′)₂ fragment of a humanized anti-CD40antibody comprising a heavy chain variable domain amino acid sequenceand a light chain variable domain amino acid sequence of SEQ ID NO:7 andSEQ ID NO:14, respectively; SEQ ID NO:6 and SEQ ID NO:16, respectively;SEQ ID NO:7 and SEQ ID NO:16, respectively; SEQ ID NO:10 and SEQ IDNO:14, respectively; SEQ ID NO:11 and SEQ ID NO:14, respectively; SEQ IDNO:10 and SEQ ID NO:16, respectively; and SEQ ID NO: 11 and SEQ IDNO:16, respectively.

Yet other embodiments include a F(ab′)₂ fragment of a humanizedanti-CD40 antibody comprising a heavy chain variable domain amino acidsequence and a light chain variable domain amino acid sequence of SEQ IDNO:7 and SEQ ID NO:14, respectively; SEQ ID NO:6 and SEQ ID NO:16,respectively; SEQ ID NO:10 and SEQ ID NO:16, respectively; and SEQ IDNO: 11 and SEQ ID NO:16, respectively.

Some embodiments include a F(ab′)₂ fragment of a humanized anti-CD40antibody that contains a heavy chain variable domain comprising theamino acid sequence of SEQ ID NO:10 and a light chain variable domaincomprising the amino acid sequence of SEQ ID NO:16.

In some embodiments, the antibody or antibody fragment includes aconstant region that mediates effector function. The constant region canprovide antibody-dependent cellular cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP) and/orcomplement-dependent cytotoxicity (CDC) responses against aCD40-expressing target cell. The effector domain(s) can be, for example,an Fc region of an Ig molecule. Typically, the CD40 binding agentrecruits and/or activates cytotoxic white blood cells (e.g., naturalkiller (NK) cells, phagocytotic cells (e.g., macrophages), and/or serumcomplement components).

The effector domain of an antibody can be from any suitable vertebrateanimal species and isotypes. The isotypes from different animal speciesdiffer in the abilities to mediate effector functions. For example, theability of human immunoglobulin to mediate CDC and ADCC/ADCP isgenerally in the order of IgM≈IgG₁≈IgG₃>IgG₂>IgG₄ andIgG₁≈IgG₃>IgG₂/IgM/IgG₄, respectively. Murine immunoglobulins mediateCDC and ADCC/ADCP generally in the order of murineIgM≈IgG₃>>IgG_(2b)>IgG_(2a)>>IgG₁ and IgG_(2b)>IgG₂a>IgG₁>>IgG₃,respectively. In another example, murine IgG_(2a) mediates ADCC whileboth murine IgG_(2a) and IgM mediate CDC.

Antibody Modifications

The humanized anti-CD40 antibodies and agents can include modificationsof the humanized anti-CD40 antibody or antigen-binding fragment thereof.For example, it may be desirable to modify the antibody with respect toeffector function, so as to enhance the effectiveness of the antibody intreating cancer, for example. One such modification is the introductionof cysteine residue(s) into the Fc region, thereby allowing interchaindisulfide bond formation in this region. The homodimeric antibody thusgenerated can have improved internalization capability and/or increasedcomplement-mediated cell killing and/or antibody-dependent cellularcytotoxicity (ADCC). See, for example, Caron et al., 1992, J. Exp Med.176:1191-1195; and Shopes, 1992, J. Immunol. 148:2918-2922. Homodimericantibodies having enhanced anti-tumor activity can also be preparedusing heterobifunctional cross-linkers as described in Wolff et al.,1993, Cancer Research 53: 2560-2565. Alternatively, an antibody can beengineered to contain dual Fc regions, enhancing complement lysis andADCC capabilities of the antibody. See Stevenson et al., 1989,Anti-Cancer Drug Design 3: 219-230.

Antibodies with improved ability to support ADCC have been generated bymodifying the glycosylation pattern of their Fc region. This is possiblesince antibody glycosylation at the asparagine residue, N297, in theC_(H)2 domain is involved in the interaction between IgG and Fcγreceptors prerequisite to ADCC. Host cell lines have been engineered toexpress antibodies with altered glycosylation, such as increasedbisecting N-acetylglucosamine or reduced fucose. Fucose reductionprovides greater enhancement to ADCC activity than does increasing thepresence of bisecting N-acetylglucosamine. Moreover, enhancement of ADCCby low fucose antibodies is independent of the FcγRIIIa V/Fpolymorphism.

Modifying the amino acid sequence of the Fc region of antibodies is analternative to glycosylation engineering to enhance ADCC. The bindingsite on human IgG₁ for Fcγ receptors has been determined by extensivemutational analysis. This led to the generation of humanized IgG₁antibodies with Fc mutations that increase the binding affinity forFcγRIIIa and enhance ADCC in vitro. Additionally, Fc variants have beenobtained with many different permutations of binding properties, e.g.,improved binding to specific FcγR receptors with unchanged or diminishedbinding to other FcγR receptors.

In some embodiments, the Fc region can be modified as described in U.S.Patent Application Publication Nos. 2006-0003412 and 2006-0008883, thedisclosures of which are incorporated by reference herein.

Another aspect includes immunoconjugates comprising the humanizedantibody or fragments thereof conjugated to a cytotoxic agent such as achemotherapeutic agent, a toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used to form useful immunoconjugatesinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, the tricothecenes, and the like. A variety of radionuclidesare available for the production of radioconjugated humanized anti-CD40antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the humanized anti-CD40 antibody and cytotoxic orchemotherapeutic agent can be made by known methods, using a variety ofbifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., 1987, Science 238:1098.Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See, e.g., InternationalPublication WO 94/11026. Conjugates also can be formed with a cleavablelinker, such as that disclosed in published EP Patent Application 0 624377; the disclosure of which is incorporated by reference herein.

In another embodiment, the antibody may be conjugated to a “receptor”(such as streptavidin) for utilization in tumor pretargeting. In thisprocedure, the antibody-receptor conjugate is administered to a patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” that selectivelybinds the receptor (e.g., avidin), the ligand being conjugated to acytotoxic agent (e.g., a radionuclide).

The humanized anti-CD40 antibodies disclosed herein can also beformulated as immunoliposomes. Liposomes containing the antibody areprepared by methods known in the art, such as described in Epstein etal., 1985, Proc. Natl. Acad. Sci. USA 82:3688; Hwang et al., 1980, Proc.Natl. Acad. Sci. USA 77:4030; and U.S. Pat. Nos. 4,485,045 and4,544,545. Liposomes having enhanced circulation time are disclosed, forexample, in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of an antibody disclosed herein can beconjugated to the liposomes as described in Martin et al., 1982, J.Biol. Chem. 257:286-288 via a disulfide interchange reaction. Achemotherapeutic agent (such as doxorubicin) is optionally containedwithin the liposome. See, e.g., Gabizon et al., 1989, J. National CancerInst. 81(19):1484.

The antibodies described and disclosed herein can also be used in ADEPT(Antibody-Directed Enzyme Prodrug Therapy) procedures by conjugating theantibody to a prodrug-activating enzyme that converts a prodrug (e.g., apeptidyl chemotherapeutic agent), to an active anti-cancer drug. See,for example, WO 81/01145, WO 88/07378, and U.S. Pat. No. 4,975,278. Theenzyme component of the immunoconjugate useful for ADEPT is an enzymecapable of acting on a prodrug in such a way so as to covert it into itsmore active, cytotoxic form. Specific enzymes that are useful in ADEPTinclude, but are not limited to, alkaline phosphatase for convertingphosphate-containing prodrugs into free drugs; arylsulfatase forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase for converting non-toxic 5-fluorocytosine into the anti-cancerdrug, 5-fluorouracil; proteases, such as serratia protease, thermolysin,subtilisin, carboxypeptidases, and cathepsins (such as cathepsins B andL), for converting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, for converting prodrugs containing D-aminoacid substituents; carbohydrate-cleaving enzymes such as β-galactosidaseand neuraminidase for converting glycosylated prodrugs into free drugs;β-lactamase for converting drugs derivatized with β-lactams into freedrugs; and penicillin amidases, such as penicillin V amidase orpenicillin G amidase, for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies having enzymatic activity(“abzymes”) can be used to convert the prodrugs into free active drugs(see, for example, Massey, 1987, Nature 328: 457-458). Antibody-abzymeconjugates can be prepared by known methods for delivery of the abzymeto a tumor cell population, for example, by covalently binding theenzyme to the humanized anti-CD40 antibody/heterobifunctionalcrosslinking reagents discussed above. Alternatively, fusion proteinscomprising at least the antigen binding region of an antibody disclosedherein linked to at least a functionally active portion of an enzyme asdescribed above can be constructed using recombinant DNA techniques(see, e.g., Neuberger et al., 1984, Nature 312:604-608).

In certain embodiments, it may be desirable to use a humanized anti-CD40antibody fragment, rather than an intact antibody, to increase tumorpenetration, for example. It may be desirable to modify the antibodyfragment in order to increase its serum half life. This can be achieved,for example, by incorporation of a salvage receptor binding epitope intothe antibody fragment. In one method, the appropriate region of theantibody fragment can be altered (e.g., mutated), or the epitope can beincorporated into a peptide tag that is then fused to the antibodyfragment at either end or in the middle, for example, by DNA or peptidesynthesis. See, e.g., WO 96/32478.

In other embodiments, covalent modifications of the humanized anti-CD40antibody are also included. They may be made by chemical synthesis or byenzymatic or chemical cleavage of the antibody, if applicable. Othertypes of covalent modifications of the antibody can be introduced intothe molecule by reacting targeted amino acid residues of the antibodywith an organic derivatizing agent that is capable of reacting withselected side chains or the amino- or carboxy-terminal residues.

Covalent modifications include modification of cysteinyl residues,histidyl residues, lysinyl and amino-terminal residues, arginylresidues, tyrosyl residues, carboxyl side groups (aspartyl or glutamyl),glutaminyl and asparaginyl residues, or seryl, or threonyl residues.Another type of covalent modification involves chemically orenzymatically coupling glycosides to the antibody.

Removal of any carbohydrate moieties present on the antibody can beaccomplished chemically or enzymatically. Chemical deglycosylation isdescribed by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 andby Edge et al., 1981, Anal. Biochem., 118:131. Enzymatic cleavage ofcarbohydrate moieties on antibodies can be achieved by the use of avariety of endo- and exo-glycosidases as described by Thotakura et al.,1987, Meth. Enzymol. 138:350.

Another type of useful covalent modification comprises linking theantibody to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in one or more of U.S. Pat. No. 4,640,835, U.S. Pat.No. 4,496,689, U.S. Pat. No. 4,301,144, U.S. Pat. No. 4,670,417, U.S.Pat. No. 4,791,192 and U.S. Pat. No. 4,179,337.

Humanization and Amino Acid Sequence Variants

Example 1 below describes procedures for humanization of an anti-CD40antibody while Example 2 describes variants. In certain embodiments, itmay be desirable to generate amino acid sequence variants of thesehumanized antibodies, particularly where these improve the bindingaffinity or other biological properties of the humanized antibody.

Amino acid sequence variants of the anti-CD40 antibody can be preparedby introducing appropriate nucleotide changes into the anti-CD40antibody DNA, or by peptide synthesis. Such variants include, forexample, deletions from, and/or insertions into and/or substitutions of,residues within the amino acid sequences of the anti-CD40 antibodies ofthe examples herein. Any combination of deletions, insertions, andsubstitutions is made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics. The aminoacid changes also may alter post-translational processes of thehumanized or variant anti-CD40 antibody, such as changing the number orposition of glycosylation sites.

A useful method for identification of certain residues or regions of theanti-CD40 antibody that are preferred locations for mutagenesis iscalled “alanine scanning mutagenesis,” as described by Cunningham andWells (Science, 244:1081-1085 (1989)). Here, a residue or group oftarget residues are identified (e.g., charged residues such as arg, asp,his, lys, and glu) and replaced by a neutral or negatively charged aminoacid (typically alanine) to affect the interaction of the amino acidswith CD40 antigen. Those amino acid locations demonstrating functionalsensitivity to the substitutions then are refined by introducing furtheror other variants at, or for, the sites of substitution. Thus, while thesite for introducing an amino acid sequence variation is predetermined,the nature of the mutation per se need not be predetermined. Forexample, to analyze the performance of a mutation at a given site,alanine scanning or random mutagenesis is conducted at the target codonor region and the expressed anti-CD40 antibody variants are screened forthe desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean anti-CD40 antibody fused to an epitope tag. Other insertionalvariants of the anti-CD40 antibody molecule include a fusion to the N-or C-terminus of the anti-CD40 antibody of an enzyme or a polypeptidewhich increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the anti-CD40 antibodymolecule removed and a different residue inserted in its place. Thesites of greatest interest for substitutional mutagenesis include thehypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in Table 1 under the heading of“preferred substitutions”. If such substitutions result in a change inbiological activity, then more substantial changes, denominated“exemplary substitutions”, or as further described below in reference toamino acid classes, may be introduced and the products screened.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;asp, lys; gln arg Asp (D) glu; asn glu Cys (C) ser; ala ser Gln (Q) asn;glu asn Glu (E) asp; gln asp Gly (G) ala ala His (H) asn; gln; lys; argarg Ile (I) leu; val; met; ala; leu phe; norleucine Leu (L) norleucine;ile; val; ile met; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe;ile leu Phe (F) leu; val; ile; ala; tyr tyr Pro (P) ala ala Ser (S) thrthr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser pheVal (V) ile; leu; met; phe; leu ala; norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

-   -   (1) hydrophobic: norleucine, met, ala, val, leu, ile;    -   (2) neutral hydrophilic: cys, ser, thr;    -   (3) acidic: asp, glu;    -   (4) basic: asn, gin, his, lys, arg;    -   (5) residues that influence chain orientation: gly, pro; and    -   (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the humanized or variant anti-CD40 antibody also may be substituted,generally with serine, to improve the oxidative stability of themolecule, prevent aberrant crosslinking, or provide for establishedpoints of conjugation to a cytotoxic or cytostatic compound. Conversely,cysteine bond(s) may be added to the antibody to improve its stability(particularly where the antibody is an antibody fragment such as an Fvfragment).

A type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g., a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have improved biological properties relative tothe parent antibody from which they are generated. A convenient way forgenerating such substitutional variants is affinity maturation usingphage display. Briefly, several hypervariable region sites (e.g., 6-7sites) are mutated to generate all possible amino substitutions at eachsite. The antibody variants thus generated are displayed in a monovalentfashion from filamentous phage particles as fusions to the gene IIIproduct of M13 packaged within each particle. The phage-displayedvariants are then screened for their biological activity (e.g., bindingaffinity). In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or in addition, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and human CD40. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By “altering” is meant deletingone or more carbohydrate moieties found in the antibody, and/or addingone or more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of theanti-CD40 antibody are prepared by a variety of methods known in theart. These methods include, but are not limited to, isolation from anatural source (in the case of naturally occurring amino acid sequencevariants) or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of the anti-CD40 antibody.

Human Antibodies

As an alternative to humanization, human antibodies can be generated.For example, transgenic animals (e.g., mice) can be used that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. As an alternative tohumanization, human antibodies can be generated. For example, transgenicanimals (e.g., mice) can be used that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production. For example, it has been describedthat the homozygous deletion of the antibody heavy-chain joining region(J_(H)) gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551;Jakobovits et al., 1993, Nature 362:255-258; Bruggermann et al., 1993,Year in Immuno. 7:33; and U.S. Pat. Nos. 5,591,669; 5,589,369;5,545,807; 6,075,181; 6,150,584; 6,657,103; and 6,713,610.

Alternatively, phage display technology (see, e.g., McCafferty et al.,1990, Nature 348:552-553) can be used to produce human antibodies andantibody fragments in vitro, from immunoglobulin variable (V) domaingene repertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson and Chiswell,1993, Current Opinion in Structural Biology 3:564-571. Several sourcesof V-gene segments can be used for phage display. Clackson et al., 1991,Nature 352:624-628 isolated a diverse array of anti-oxazolone antibodiesfrom a small random combinatorial library of V genes derived from thespleens of immunized mice. A repertoire of V genes from unimmunizedhuman donors can be constructed and antibodies to a diverse array ofantigens (including self-antigens) can be isolated essentially followingthe techniques described by Marks et al., 1991, J. Mol. Biol.222:581-597, or Griffith et al., 1993, EMBO J. 12:725-734. See also U.S.Pat. Nos. 5,565,332 and 5,573,905. As discussed above, human antibodiesmay also be generated by in vitro activated B cells (see U.S. Pat. Nos.5,567,610 and 5,229,275).

Polynucleotides, Vectors, Host Cells, and Recombinant Methods

Other embodiments encompass isolated polynucleotides that comprise asequence encoding a humanized anti-CD40 antibody, vectors, and hostcells comprising the polynucleotides, and recombinant techniques forproduction of the humanized antibody. The isolated polynucleotides canencode any desired form of the anti-CD40 antibody including, forexample, full length monoclonal antibodies, Fab, Fab′, F(ab′)₂, and Fvfragments, diabodies, linear antibodies, single-chain antibodymolecules, and multispecific antibodies formed from antibody fragments.

Some embodiments include isolated polynucleotides comprising sequencesthat encode an antibody or antibody fragment having the heavy chainvariable region amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,or SEQ ID NO:11. Some embodiments include isolated polynucleotidescomprising sequences that encode an antibody or antibody fragment havingthe light chain variable domain amino acid sequence of SEQ ID NO:14, SEQID NO:15, or SEQ ID NO:16.

In one aspect, the isolated polynucleotide sequence(s) encodes anantibody or antibody fragment having a heavy chain variable domain and alight chain variable region comprising the amino acid sequences of SEQID NO:3 and SEQ ID NO:14, respectively; SEQ ID NO:4 and SEQ ID NO:14,respectively; SEQ ID NO:5 and SEQ ID NO:14, respectively; SEQ ID NO:6and SEQ ID NO:14, respectively; SEQ ID NO:7 and SEQ ID NO:14,respectively; SEQ ID NO:8 and SEQ ID NO:14, respectively; SEQ ID NO:9and SEQ ID NO:14, respectively; SEQ ID NO:6 and SEQ ID NO:15,respectively; SEQ ID NO:6 and SEQ ID NO:16, respectively; SEQ ID NO:7and SEQ ID NO:16, respectively; SEQ ID NO:10 and SEQ ID NO:14,respectively; SEQ ID NO:11 and SEQ ID NO:14, respectively; SEQ ID NO:10and SEQ ID NO:16, respectively; or SEQ ID NO:11 and SEQ ID NO:16,respectively.

In another aspect, the isolated polynucleotide sequence(s) encodes anantibody or antibody fragment having a heavy chain variable domain and alight chain variable domain comprising the amino acid sequences of SEQID NO:7 and SEQ ID NO:14, respectively; SEQ ID NO:6 and SEQ ID NO:16,respectively; SEQ ID NO:7 and SEQ ID NO:16, respectively; SEQ ID NO:10and SEQ ID NO:14, respectively; SEQ ID NO:11 and SEQ ID NO:14,respectively; SEQ ID NO:10 and SEQ ID NO:16, respectively; and SEQ IDNO: 11 and SEQ ID NO:16, respectively.

In yet another aspect, the isolated polynucleotide sequence(s) encodesan antibody or antibody fragment having a heavy chain variable domainand a light chain variable domain comprising the amino acid sequences ofSEQ ID NO:7 and SEQ ID NO:14, respectively; SEQ ID NO:6 and SEQ IDNO:16, respectively; SEQ ID NO:10 and SEQ ID NO:16, respectively; andSEQ ID NO: 11 and SEQ ID NO:16, respectively.

In yet another aspect, the isolated polynucleotide sequence(s) encodesan antibody or antibody fragment having a heavy chain variable domainand a light chain variable domain comprising the amino acid sequences ofSEQ ID NO:10 and a light chain variable domain comprising the amino acidsequence of SEQ ID NO:16.

The polynucleotide(s) that comprise a sequence encoding a humanizedanti-CD40 antibody or a fragment or chain thereof can be fused to one ormore regulatory or control sequence, as known in the art, and can becontained in suitable expression vectors or host cell as known in theart. Each of the polynucleotide molecules encoding the heavy or lightchain variable domains can be independently fused to a polynucleotidesequence encoding a constant domain, such as a human constant domain,enabling the production of intact antibodies. Alternatively,polynucleotides, or portions thereof, can be fused together, providing atemplate for production of a single chain antibody.

For recombinant production, a polynucleotide encoding the antibody isinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Many suitable vectors for expressing the recombinantantibody are available. The vector components generally include, but arenot limited to, one or more of the following: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence.

The humanized anti-CD40 antibodies can also be produced as fusionpolypeptides, in which the antibody is fused with a heterologouspolypeptide, such as a signal sequence or other polypeptide having aspecific cleavage site at the amino terminus of the mature protein orpolypeptide. The heterologous signal sequence selected is typically onethat is recognized and processed (i.e., cleaved by a signal peptidase)by the host cell. For prokaryotic host cells that do not recognize andprocess the humanized anti-CD40 antibody signal sequence, the signalsequence can be substituted by a prokaryotic signal sequence. The signalsequence can be, for example, alkaline phosphatase, penicillinase,lipoprotein, heat-stable enterotoxin II leaders, and the like. For yeastsecretion, the native signal sequence can be substituted, for example,with a leader sequence obtained from yeast invertase alpha-factor(including Saccharomyces and Kluyveromyces α-factor leaders), acidphosphatase, C. albicans glucoamylase, or the signal described inWO90/13646. In mammalian cells, mammalian signal sequences as well asviral secretory leaders, for example, the herpes simplex gD signal, canbe used. The DNA for such precursor region is ligated in reading frameto DNA encoding the humanized anti-CD40 antibody.

Expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV, andBPV) are useful for cloning vectors in mammalian cells. Generally, theorigin of replication component is not needed for mammalian expressionvectors (the SV40 origin may typically be used only because it containsthe early promoter).

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid, and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up anucleic acid encoding a humanized anti-CD40 antibody, such as DHFR(dihydrofolate reductase), thymidine kinase, metallothionein-I and -II(such as primate metallothionein genes), adenosine deaminase, ornithinedecarboxylase, and the like.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity (e.g., DG44).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding anti-CD40 antibody, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH),can be selected by cell growth in medium containing a selection agentfor the selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See, e.g., U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the TRP1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., 1979, Nature 282: 39). TheTRP1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1 (Jones, 1977, Genetics 85:12). The presence of the trp1 lesionin the yeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.Similarly, Leu2p-deficient yeast strains such as ATCC 20,622 and 38,626are complemented by known plasmids bearing the LEU2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis (Van den Berg, 1990, Bio/Technology8:135). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed (Fleer et al., 1991, Bio/Technology 9:968-975).

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid molecule encoding an anti-CD40 antibody or polypeptide chainthereof. Promoters suitable for use with prokaryotic hosts include phoApromoter, β-lactamase and lactose promoter systems, alkalinephosphatase, tryptophan (trp) promoter system, and hybrid promoters suchas the tac promoter. Other known bacterial promoters are also suitable.Promoters for use in bacterial systems also will contain a Shine-Dalgamo(S.D.) sequence operably linked to the DNA encoding the humanizedanti-CD40 antibody.

Many eukaryotic promoter sequences are known. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Inducible promoters have the additional advantage of transcriptioncontrolled by growth conditions. These include yeast promoter regionsfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,derivative enzymes associated with nitrogen metabolism, metallothionein,glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible formaltose and galactose utilization. Suitable vectors and promoters foruse in yeast expression are further described in EP 73,657. Yeastenhancers also are advantageously used with yeast promoters.

Humanized anti-CD40 antibody transcription from vectors in mammalianhost cells is controlled, for example, by promoters obtained from thegenomes of viruses such as polyoma virus, fowlpox virus, adenovirus(such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, or from heat-shock promoters, providedsuch promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., 1982, Nature 297:598-601, disclosingexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the rous sarcoma virus long terminal repeat can be used as the promoter.

Transcription of a DNA encoding a humanized anti-CD40 antibody disclosedherein by higher eukaryotes is often increased by inserting an enhancersequence into the vector. Many enhancer sequences are now known frommammalian genes (e.g., globin, elastase, albumin, α-fetoprotein, andinsulin). Typically, however, an enhancer from a eukaryotic cell virusis used. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, 1982, Nature 297:17-18for a description of enhancing elements for activation of eukaryoticpromoters. The enhancer may be spliced into the vector at a position 5′or 3′ to the humanized anti-CD40 antibody-encoding sequence, but ispreferably located at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) can also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding anti-CD40 antibody. One usefultranscription termination component is the bovine growth hormonepolyadenylation region. See WO94/11026 and the expression vectordisclosed therein. In some embodiments, humanized anti-CD40 antibodiescan be expressed using the CHEF system. (See, e.g., U.S. Pat. No.5,888,809; the disclosure of which is incorporated by reference herein.)

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for humanizedanti-CD40 antibody-encoding vectors. Saccharomyces cerevisiae, or commonbaker's yeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastors (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated humanizedanti-CD40 antibody are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells, including, e.g.,numerous baculoviral strains and variants and corresponding permissiveinsect host cells from hosts such as Spodoptera frugiperda(caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito),Drosophila melanogaster (fruitfly), and Bombyx mori (silk worm). Avariety of viral strains for transfection are publicly available, e.g.,the L-1 variant of Autographa californica NPV and the Bm-5 strain ofBombyx mori NPV, and such viruses may be used, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can also be utilized as hosts.

In another aspect, expression of humanized anti-CD40 is carried out invertebrate cells. The propagation of vertebrate cells in culture (tissueculture) has become routine procedure and techniques are widelyavailable. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651), humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, (Graham et al., 1977, J. Gen Virol. 36: 59), babyhamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovarycells/-DHFR⁻ (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216; e.g., DG44), mouse sertoli cells (TM4, Mather, 1980, Biol. Reprod.23:243-251), monkey kidney cells (CV1 ATCC CCL 70), African green monkeykidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells(HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo ratliver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT060562, ATCC CCL51), TRI cells (Mather et al., 1982, Annals N.Y. Acad.Sci. 383: 44-68), MRC 5 cells, FS4 cells, and human hepatoma line (HepG2).

Host cells are transformed with the above-described expression orcloning vectors for humanized anti-CD40 antibody production and culturedin conventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

The host cells used to produce a humanized anti-CD40 antibody describedherein may be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma-Aldrich Co., St. Louis, Mo.), MinimalEssential Medium ((MEM), (Sigma-Aldrich Co.), RPMI-1640 (Sigma-AldrichCo.), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma-Aldrich Co.)are suitable for culturing the host cells. In addition, any of the mediadescribed in one or more of Ham et al., 1979, Meth. Enz. 58: 44, Barneset al., 1980, Anal. Biochem. 102: 255, U.S. Pat. No. 4,767,704, U.S.Pat. No. 4,657,866, U.S. Pat. No. 4,927,762, U.S. Pat. No. 4,560,655,U.S. Pat. No. 5,122,469, WO 90/103430, and WO 87/00195 may be used asculture media for the host cells. Any of these media may be supplementedas necessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleotides (such as adenosine and thymidine), antibiotics (such asgentamicin), trace elements (defined as inorganic compounds usuallypresent at final concentrations in the micromolar range), and glucose oran equivalent energy source. Other supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, the cells may bedisrupted to release protein as a first step. Particulate debris, eitherhost cells or lysed fragments, can be removed, for example, bycentrifugation or ultrafiltration. Carter et al., 1992, Bio/Technology10:163-167 describes a procedure for isolating antibodies that aresecreted to the periplasmic space of E. coli. Briefly, cell paste isthawed in the presence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 minutes. Cell debriscan be removed by centrifugation. Where the antibody is secreted intothe medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants. A variety of methodscan be used to isolate the antibody from the host cell.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing a typical purification technique. The suitability of protein A asan affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human gamma1, gamma2, orgamma4 heavy chains (see, e.g., Lindmark et al., 1983 J. Immunol. Meth.62:1-13). Protein G is recommended for all mouse isotypes and for humangamma3 (see, e.g., Guss et al., 1986 EMBO J. 5:1567-1575). A matrix towhich an affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe antibody comprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, reverse phase HPLC, chromatography onsilica, chromatography on heparin SEPHAROSE™ chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, typically performed at low salt concentrations(e.g., from about 0-0.25M salt).

Hybridization Conditions

Also included are nucleic acids that hybridize under low, moderate, andhigh stringency conditions, as defined herein, to all or a portion(e.g., the portion encoding the variable region) of the nucleotidesequence represented by SEQ ID NO:17, or its complement, or SEQ ID NO:20or its complement. The hybridizing portion of the hybridizing nucleicacid is typically at least 15 (e.g., 20, 25, 30 or 50) nucleotides inlength. The hybridizing portion of the hybridizing nucleic acid is atleast 80%, e.g., at least 90%, at least 95%, or at least 98%, identicalto the sequence of a portion or all of a nucleic acid encoding ananti-CD40 polypeptide (e.g., a heavy chain or light chain variableregion), or its complement. Hybridizing nucleic acids of the typedescribed herein can be used, for example, as a cloning probe, a primer,e.g., a PCR primer, or a diagnostic probe.

By way of example and not limitation, procedures using conditions of lowstringency are as follows (see also Shilo and Weinberg, 1981, Proc.Natl. Acad. Sci. USA 78:6789-6792). In one embodiment, filterscontaining DNA are pretreated for 6 h at 40° C. in a solution containing35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1%Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizationsare carried out in the same solution with the following modifications:0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10%(wt/vol) dextran sulfate, and 5-20×10⁶ cpm ³²P-labeled probe is used.Filters are incubated in hybridization mixture for 18-20 h at 40° C.,and then washed for 1.5 h at 55° C. in a solution containing 2×SSC, 25mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution isreplaced with fresh solution and incubated an additional 1.5 h at 60° C.Filters are blotted dry and exposed for autoradiography. If necessary,filters are washed for a third time at 65-68° C. and re-exposed to film.In another embodiment, an example of low stringency conditions includeshybridization in a buffer comprising 35% formamide, 5×SSC, 50 mMTris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100μg/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate, for18-20 hours at 40° C., washing in a buffer consisting of 2×SSC, 25 mMTris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS, for 1.5 hours at 55° C., andwashing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mMEDTA, and 0.1% SDS, for 1.5 hours at 60° C. Other conditions of lowstringency that may be used are well known in the art (e.g., as employedfor cross-species hybridizations).

By way of example and not limitation, procedures using conditions ofhigh stringency are as follows. Prehybridization of filters containingDNA is carried out for 8 h to overnight at 65° C. in buffer composed of6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll,0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters arehybridized for 48 h at 65° C. in prehybridization mixture containing 100μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe.Washing of filters is done at 37° C. for 1 h in a solution containing2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by awash in 0.1×SSC at 50° C. for 45 min before autoradiography. Otherconditions of high stringency that may be used are well known in theart.

By way of example and not limitation, procedures using conditions ofmoderate stringency are as follows: Filters containing DNA arepretreated for 6 hours at 55° C. in a solution containing 6×SSC,5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon spermDNA. Hybridizations are carried out in the same solution with 5-20×10⁶cpm ³²P-labeled probe. Filters are incubated in hybridization mixturefor 18-20 hours at 55° C., and then washed twice for 30 minutes at 60°C. in a solution containing 1×SSC and 0.1% SDS. Filters are blotted dryand exposed for autoradiography. Washing of filters is done at 37° C.for 1 hour in a solution containing 2×SSC, 0.1% SDS. Other conditions ofmoderate stringency that may be used are well-known in the art (see,e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2dEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Sambrook et al., 2001; Molecular Cloning, A Laboratory Manual, 3rd ed.,Cold Spring Harbor Publish., Cold Spring Harbor, N.Y.; see also Ausubelet al., eds., in Current Protocols in Molecular Biology series oflaboratory technique manuals, 1987-1999, Current Protocols,© 1994-199John Wiley and Sons, Inc.).

Some embodiments include isolated polynucleotides including sequencesthat encode an antibody or antibody fragment having the heavy chainvariable region amino acid sequence that is at least 80%, at least 90%,at least 95%, at least 98%, or at least 99% identical to the amino acidsequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11. Someembodiments include isolated polynucleotides including sequences thatencode an antibody or antibody fragment having the light chain variabledomain amino acid sequence that is at least 80%, at least 90%, at least95%, at least 98%, or at least 99% identical to the amino acid sequenceof SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16.

In one aspect, the isolated polynucleotide sequence(s) encodes anantibody or antibody fragment having a heavy chain variable domain and alight chain variable region, each including an amino acid sequence thatis at least 80%, at least 90%, at least 95%, at least 98%, or at least99% identical to the amino acid sequence of SEQ ID NO:3 and SEQ IDNO:14, respectively; SEQ ID NO:4 and SEQ ID NO:14, respectively; SEQ IDNO:5 and SEQ ID NO:14, respectively; SEQ ID NO:6 and SEQ ID NO:14,respectively; SEQ ID NO:7 and SEQ ID NO:14, respectively; SEQ ID NO:8and SEQ ID NO:14, respectively; SEQ ID NO:9 and SEQ ID NO:14,respectively; SEQ ID NO:6 and SEQ ID NO:15, respectively; SEQ ID NO:6and SEQ ID NO:16, respectively; SEQ ID NO:7 and SEQ ID NO:16,respectively; SEQ ID NO:10 and SEQ ID NO:14, respectively; SEQ ID NO:11and SEQ ID NO:14, respectively; SEQ ID NO:10 and SEQ ID NO:16,respectively; or SEQ ID NO:11 and SEQ ID NO:16, respectively.

In another aspect, the isolated polynucleotide sequence(s) encodes anantibody or antibody fragment having a heavy chain variable domain and alight chain variable domain, each including an amino acid sequence thatis at least 80%, at least 90%, at least 95%, at least 98%, or at least99% identical to the amino acid sequence of SEQ ID NO:7 and SEQ IDNO:14, respectively; SEQ ID NO:6 and SEQ ID NO:16, respectively; SEQ IDNO:7 and SEQ ID NO:16, respectively; SEQ ID NO:10 and SEQ ID NO:14,respectively; SEQ ID NO:11 and SEQ ID NO:14, respectively; SEQ ID NO:10and SEQ ID NO:16, respectively; and SEQ ID NO: 11 and SEQ ID NO:16,respectively.

In yet another aspect, the isolated polynucleotide sequence(s) encodesan antibody or antibody fragment having a heavy chain variable regionand a light chain variable region, each including an amino acid sequencethat is at least 80%, at least 90%, at least 95%, at least 98%, or atleast 99% identical to the amino acid sequence of SEQ ID NO:7 and SEQ IDNO:14, respectively; SEQ ID NO:6 and SEQ ID NO:16, respectively; SEQ IDNO:10 and SEQ ID NO:16, respectively; and SEQ ID NO: 11 and SEQ IDNO:16, respectively.

In yet another aspect, the isolated polynucleotide sequence(s) encodesan antibody or antibody fragment having a heavy chain variable regionand a light chain variable region, each including an amino acid sequencethat is at least 80%, at least 90%, at least 95%, at least 98%, or atleast 99% identical to the amino acid sequence of SEQ ID NO:10 and SEQID NO:16, respectively.

As used herein, the terms “identical” or “percent identity,” in thecontext of two or more nucleic acids or polypeptide sequences, refer totwo or more sequences or subsequences that are the same or have aspecified percentage of nucleotides or amino acid residues that are thesame, when compared and aligned for maximum correspondence. To determinethe percent identity, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoor nucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In some embodiments, the two sequences that arecompared are the same length after gaps are introduced within thesequences, as appropriate (e.g., excluding additional sequence extendingbeyond the sequences being compared). For example, when variable regionsequences are compared, the leader and/or constant domain sequences arenot considered. For sequence comparisons between two sequences, a“corresponding” CDR refers to a CDR in the same location in bothsequences (e.g., CDR-H1 of each sequence).

The determination of percent identity or percent similarity between twosequences can be accomplished using a mathematical algorithm. Apreferred, non-limiting example of a mathematical algorithm utilized forthe comparison of two sequences is the algorithm of Karlin and Altschul,1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin andAltschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12, to obtain nucleotide sequences homologous to a nucleicacid encoding a protein of interest. BLAST protein searches can beperformed with the XBLAST program, score=50, wordlength=3, to obtainamino acid sequences homologous to protein of interest. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules (Id.). When utilizingBLAST, Gapped BLAST, and PSI-Blast programs, the default parameters ofthe respective programs (e.g., XBLAST and NBLAST) can be used. Anotherpreferred, non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Myers and Miller, CABIOS(1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM 120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. Additional algorithms for sequenceanalysis are known in the art and include ADVANCE and ADAM as describedin Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTAdescribed in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA85:2444-8. Within FASTA, ktup is a control option that sets thesensitivity and speed of the search. If ktup=2, similar regions in thetwo sequences being compared are found by looking at pairs of alignedresidues; if ktup=1, single aligned amino acids are examined. ktup canbe set to 2 or 1 for protein sequences, or from 1 to 6 for DNAsequences. The default if ktup is not specified is 2 for proteins and 6for DNA. Alternatively, protein sequence alignment may be carried outusing the CLUSTAL W algorithm, as described by Higgins et al., 1996,Methods Enzymol. 266:383-402.

Non-Therapeutic Uses

The antibodies described herein are useful as affinity purificationagents. In this process, the antibodies are immobilized on a solid phasesuch a Sephadex resin or filter paper, using methods well known in theart. The immobilized antibody is contacted with a sample containing theCD40 protein (or fragment thereof) to be purified, and thereafter thesupport is washed with a suitable solvent that will remove substantiallyall the material in the sample except the CD40 protein, which is boundto the immobilized antibody. Finally, the support is washed with anothersuitable solvent, such as glycine buffer, pH 5.0 that will release theCD40 protein from the antibody.

Humanized anti-CD40 antibodies are also useful in diagnostic assays todetect and/or quantify CD40 protein, for example, detecting CD40expression in specific cells, tissues, or serum.

For diagnostic applications, the antibody typically will be labeled witha detectable moiety. Numerous labels are available which can begenerally grouped into the following categories:

-   -   (a) Radioisotopes, for example, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and        ¹³¹I. The antibody can be labeled with the radioisotope, using        the techniques described in, for example, Current Protocols in        Immunology, Volumes 1 and 2, 1991, Coligen et al., Ed.        Wiley-Interscience, New York, N.Y., Pubs. Radioactivity can be        measured, for example, by scintillation counting.    -   (b) Fluorescent labels such as rare earth chelates (europium        chelates) or fluorescein and its derivatives, rhodamine and its        derivatives, dansyl, Lissamine, phycoerythrin, and Texas Red are        available. The fluorescent labels can be conjugated to the        antibody via known techniques, such as those disclosed in        Current Protocols in Immunology, supra, for example.        Fluorescence can be quantified using a fluorimeter.    -   (c) Various enzyme-substrate labels are available (see, e.g.,        U.S. Pat. No. 4,275,149 provides a review of some of these). The        enzyme generally catalyzes a chemical alteration of the        chromogenic substrate that can be measured using various        techniques. For example, the enzyme may catalyze a color change        in a substrate that can be measured spectrophotometrically.        Alternatively, the enzyme may alter the fluorescence or        chemiluminescence of the substrate. Techniques for quantifying a        change in fluorescence are described above. The chemiluminescent        substrate becomes electronically excited by a chemical reaction        and may then emit light that can be measured, using a        chemiluminometer, for example, or donates energy to a        fluorescent acceptor. Examples of enzymatic labels include        luciferases such as firefly luciferase and bacterial luciferase        (U.S. Pat. No. 4,737,456), luciferin,        2,3-dihydrophthalazinediones, malate dehydrogenase, urease,        peroxidase such as horseradish peroxidase (HRPO), alkaline        phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide        oxidases (such as glucose oxidase, galactose oxidase, and        glucose-6-phosphate dehydrogenase), heterocydic oxidases (such        as uricase and xanthine oxidase), lactoperoxidase,        microperoxidase, and the like. Techniques for conjugating        enzymes to antibodies are described, for example, in O'Sullivan        et al., 1981, Methods for the Preparation of Enzyme-Antibody        Conjugates for use in Enzyme Immunoassay, in Methods in Enzym.        (J. Langone & H. Van Vunakis, eds.), Academic press, N.Y., 73:        147-166.

Examples of enzyme-substrate combinations include, for example:

-   -   (a) Horseradish peroxidase (HRPO) with hydrogen peroxidase as a        substrate, wherein the hydrogen peroxidase oxidizes a dye        precursor such as orthophenylene diamine (OPD) or        3,3′,5,5′-tetramethyl benzidine hydrochloride (TMB);    -   (b) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as        chromogenic substrate; and    -   (c) β-D-galactosidase (β-D-Gal) with a chromogenic substrate        such as p-nitrophenyl-β-D-galactosidase or fluorogenic substrate        4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. No.4,275,149 and U.S. Pat. No. 4,318,980.

The label may be indirectly conjugated with the antibody using variousknown techniques. For example, the antibody can be conjugated withbiotin and any of the three broad categories of labels mentioned abovecan be conjugated with avidin, or vice versa. Biotin binds selectivelyto avidin and thus, the label can be conjugated with the antibody inthis indirect manner. Alternatively, to achieve indirect conjugation ofthe label with the antibody, the antibody can be conjugated with a smallhapten (such as digoxin) and one of the different types of labelsmentioned above is conjugated with an anti-hapten antibody (e.g.,anti-digoxin antibody). Thus, indirect conjugation of the label with theantibody can be achieved.

In another embodiment, the humanized anti-CD40 antibody is usedunlabeled and detected with a labeled antibody that binds the humanizedanti-CD40 antibody.

The antibodies described herein may be employed in any known assaymethod, such as competitive binding assays, direct and indirect sandwichassays, and immunoprecipitation assays. See, e.g., Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

Diagnostic Kits

A humanized anti-CD40 antibody can be used in a diagnostic kit, i.e., apackaged combination of reagents in predetermined amounts withinstructions for performing the diagnostic assay. Where the antibody islabeled with an enzyme, the kit may include substrates and cofactorsrequired by the enzyme such as a substrate precursor that provides thedetectable chromophore or fluorophore. In addition, other additives maybe included such as stabilizers, buffers (for example a block buffer orlysis buffer), and the like. The relative amounts of the variousreagents may be varied widely to provide for concentrations in solutionof the reagents that substantially optimize the sensitivity of theassay. The reagents may be provided as dry powders, usually lyophilized,including excipients that on dissolution will provide a reagent solutionhaving the appropriate concentration.

Therapeutic Uses

In another embodiment, a humanized anti-CD40 antibody disclosed hereinis useful in the treatment of various disorders associated with theexpression of CD40 as described herein.

The humanized anti-CD40 antibody or agent is administered by anysuitable means, including parenteral, subcutaneous, intraperitoneal,intrapulmonary, and intranasal, and, if desired for localimmunosuppressive treatment, intralesional administration (includingperfusing or otherwise contacting the graft with the antibody beforetransplantation). The humanized anti-CD40 antibody or agent can beadministered, for example, as an infusion or as a bolus. Parenteralinfusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. In addition, thehumanized anti-CD40 antibody is suitably administered by pulse infusion,particularly with declining doses of the antibody. In one aspect, thedosing is given by injections, most preferably intravenous orsubcutaneous injections, depending in part on whether the administrationis brief or chronic.

For the prevention or treatment of disease, the appropriate dosage ofantibody will depend on a variety of factors such as the type of diseaseto be treated, as defined above, the severity and course of the disease,whether the antibody is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the antibody, and the discretion of the attending physician. Theantibody is suitably administered to the patient at one time or over aseries of treatments.

Depending on the type and severity of the disease, about 1 μg/kg to 20mg/kg (e.g., 0.1-15 mg/kg) of antibody is an initial candidate dosagefor administration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment is sustaineduntil a desired suppression of disease symptoms occurs. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays. An exemplary dosingregimen is that disclosed in WO 94/04188.

The antibody composition will be formulated, dosed, and administered ina fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the antibody to be administeredwill be governed by such considerations, and is the minimum amountnecessary to prevent, ameliorate, or treat the disorder associated withCD40 expression.

The antibody need not be, but is optionally, formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of humanizedanti-CD40 antibody present in the formulation, the type of disorder ortreatment, and other factors discussed above. These are generally usedin the same dosages and with administration routes as used hereinbeforeor about from 1 to 99% of the heretofore employed dosages.

CD40-Associated Disorders

The anti-CD40 antibodies or agents are useful for treating or preventinga CD40-expressing cancer or an immunological disorder characterized byexpression of CD40, e.g., by inappropriate activation of immune cells(e.g., lymphocytes or dendritic cells). Such expression of CD40 can bedue to, for example, increased CD40 protein levels on the cells surfaceand/or altered antigenicity of the expressed CD40. Treatment orprevention of the immunological disorder, according to the methodsdescribed herein, is achieved by administering to a subject in need ofsuch treatment or prevention an effective amount of the anti-CD40antibody or agent, whereby the antibody (i) binds to activated immunecells that express CD40 and that are associated with the disease stateand (ii) exerts a cytotoxic, cytostatic, or immunosuppressive effect onthe activated immune cells.

Immunological diseases that are characterized by inappropriateactivation of immune cells and that can be treated or prevented by themethods described herein can be classified, for example, by the type(s)of hypersensitivity reaction(s) that underlie the disorder. Thesereactions are typically classified into four types: anaphylacticreactions, cytotoxic (cytolytic) reactions, immune complex reactions, orcell-mediated immunity (CMI) reactions (also referred to as delayed-typehypersensitivity (DTH) reactions). (See, e.g., Fundamental Immunology(William E. Paul ed., Raven Press, N.Y., 3rd ed. 1993).)

Specific examples of such immunological diseases include the following:rheumatoid arthritis, autoimmune demyelinative diseases (e.g., multiplesclerosis, allergic encephalomyelitis), endocrine ophthalmopathy,uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Grave'sdisease, glomerulonephritis, autoimmune hepatological disorder,inflammatory bowel disease (e.g., Crohn's disease or ulcerativecolitis), anaphylaxis, allergic reaction, Sjogren's syndrome, type Idiabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis,fibromyalgia, polymyositis, dermatomyositis, inflammatory myositis,multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis,Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis,autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronichepatitis, lupoid hepatitis, atherosclerosis, subacute cutaneous lupuserythematosus, hypoparathyroidism, Dressler's syndrome, autoimmunethrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia,pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopeciaarcata, pemphigoid, scleroderma, progressive systemic sclerosis, CRESTsyndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility,sclerodactyl), and telangiectasia), male and female autoimmuneinfertility, ankylosing spondolytis, ulcerative colitis, mixedconnective tissue disease, polyarteritis nedosa, systemic necrotizingvasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome,Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrentabortion, anti-phospholipid syndrome, farmer's lung, erythemamultiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmunechronic active hepatitis, bird-fancier's lung, toxic epidermalnecrolysis, Alport's syndrome, alveolitis, allergic alveolitis,fibrosing alveolitis, interstitial lung disease, erythema nodosum,pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis,polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cellarteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema,lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome,Kawasaki's disease, dengue, encephalomyelitis, endocarditis,endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum,psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman'ssyndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis,heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy,Henoch-Schonlein purpura, graft versus host disease, transplantationrejection, cardiomyopathy, Eaton-Lambert syndrome, relapsingpolychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan'ssyndrome, acute respiratory distress syndrome, pulmonary inflammation,osteoporosis, delayed type hypersensitivity and autoimmune gonadalfailure.

Accordingly, the methods described herein encompass treatment ofdisorders of B lymphocytes (e.g., systemic lupus erythematosus,Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes),Th₁-lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis,psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave'sdisease, primary biliary cirrhosis, Wegener's granulomatosis,tuberculosis, or graft versus host disease), or Th₂-lymphocytes (e.g.,atopic dermatitis, systemic lupus erythematosus, atopic asthma,rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemicsclerosis, or chronic graft versus host disease). Generally, disordersinvolving dendritic cells involve disorders of Th₁-lymphocytes orTh₂-lymphocytes.

In some embodiments, the immunological disorder is a T cell-mediatedimmunological disorder, such as a T cell disorder in which activated Tcells associated with the disorder express CD40. Anti-CD40 antibodies oragents can be administered to deplete such CD40-expressing activated Tcells. In a specific embodiment, administration of anti-CD40 antibodiesor agents can deplete CD40-expressing activated T cells, while resting Tcells are not substantially depleted by the anti-CD40 or agent. In thiscontext, “not substantially depleted” means that less than about 60%, orless than about 70% or less than about 80% of resting T cells are notdepleted.

The anti-CD40 antibodies and agents as described herein are also usefulfor treating or preventing a CD40-expressing cancer. Treatment orprevention of a CD40-expressing cancer, according to the methodsdescribed herein, is achieved by administering to a subject in need ofsuch treatment or prevention an effective amount of the anti-CD40antibody or agent, whereby the antibody or agent (i) binds toCD40-expressing cancer cells and (ii) exerts a cytotoxic or cytostaticeffect to deplete or inhibit the proliferation of the CD40-expressingcancer cells.

CD40-expressing cancers that can be treated or prevented by the methodsdescribed herein include, for example, leukemia, such as acute leukemia,acute lymphocytic leukemia, acute myelocytic leukemia (e.g.,myeloblastic, promyelocytic, myelomonocytic, monocytic, orerythroleukemia), chronic leukemia, chronic myelocytic (granulocytic)leukemia, or chronic lymphocytic leukemia; Polycythemia vera; Lymphoma(e.g., Hodgkin's disease or Non-Hodgkin's disease); multiple myeloma,Waldenstrom's macroglobulinemia; heavy chain disease; solid tumors suchsarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,cervical cancer, uterine cancer, testicular tumor, lung carcinoma, smallcell lung carcinoma, non small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma,retinoblastoma, nasopharyngeal carcinoma, or esophageal carcinoma).

Pharmaceutical Compositions and Administration Thereof

A composition comprising a CD40 binding agent (e.g., an anti-CD40antibody) can be administered to a subject having or at risk of havingan immunological disorder or a CD40-expressing cancer. The inventionfurther provides for the use of a CD40 binding agent (e.g., an anti-CD40antibody) in the manufacture of a medicament for prevention or treatmentof a CD40 expressing cancer or immunological disorder. The term“subject” as used herein means any mammalian patient to which aCD40-binding agent can be administered, including, e.g., humans andnon-human mammals, such as primates, rodents, and dogs. Subjectsspecifically intended for treatment using the methods described hereininclude humans. The antibodies or agents can be administered eitheralone or in combination with other compositions in the prevention ortreatment of the immunological disorder or CD40-expressing cancer.

Various delivery systems are known and can be used to administer theCD40 binding agent. Methods of introduction include but are not limitedto intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, and oral routes. The CD40 bindingagent can be administered, for example by infusion, bolus or injection,and can be administered together with other biologically active agentssuch as chemotherapeutic agents. Administration can be systemic orlocal.

In specific embodiments, the CD40 binding agent composition isadministered by injection, by means of a catheter, by means of asuppository, or by means of an implant, the implant being of a porous,non-porous, or gelatinous material, including a membrane, such as asialastic membrane, or a fiber. Typically, when administering thecomposition, materials to which the anti-CD40 antibody or agent does notabsorb are used.

In other embodiments, the anti-CD40 antibody or agent is delivered in acontrolled release system. In one embodiment, a pump may be used (see,e.g., Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. RefBiomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek etal., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymericmaterials can be used. (See, e.g., Medical Applications of ControlledRelease (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974);Controlled Drug Bioavailability, Drug Product Design and Performance(Smolen and Ball eds., Wiley, New York, 1984); Ranger and Peppas, 1983,Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985,Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard etal., 1989, J. Neurosurg. 71:105.) Other controlled release systems arediscussed, for example, in Langer, supra.

A CD40 binding agent (e.g., an anti-CD40 antibody) can be administeredas pharmaceutical compositions comprising a therapeutically effectiveamount of the binding agent and one or more pharmaceutically compatibleingredients. For example, the pharmaceutical composition typicallyincludes one or more pharmaceutical carriers (e.g., sterile liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as peanut oil, soybean oil, mineral oil,sesame oil and the like). Water is a more typical carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include, for example, starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol, and thelike. Other suitable pharmaceutical excipients include amino acids(e.g., arginine, histidine, glycine), surfactants (e.g., polysorbates)and sugars and sugar alcohols (e.g., sucrose or sorbitol and otherpolyols (e.g., trehalose)). The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulations can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the nucleic acid or protein,typically in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the patient. Theformulations correspond to the mode of administration.

In typical embodiments, the pharmaceutical composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the pharmaceutical can alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the pharmaceutical is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thepharmaceutical is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients can bemixed prior to administration.

Further, the pharmaceutical composition can be provided as apharmaceutical kit comprising (a) a container containing a CD40 bindingagent (e.g., an anti-CD40 antibody) in lyophilized form and (b) a secondcontainer containing a pharmaceutically acceptable diluent (e.g.,sterile water) for injection. The pharmaceutically acceptable diluentcan be used for reconstitution or dilution of the lyophilized anti-CD40antibody or agent. Optionally associated with such container(s) can be anotice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

The amount of the CD40 binding agent (e.g., anti-CD40 antibody) that iseffective in the treatment or prevention of an immunological disorder orCD40-expressing cancer can be determined by standard clinicaltechniques. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and thestage of immunological disorder or CD40-expressing cancer, and should bedecided according to the judgment of the practitioner and each patient'scircumstances. Effective doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

For example, toxicity and therapeutic efficacy of the anti-CD40 antibodyor agent can be determined in cell cultures or experimental animals bystandard pharmaceutical procedures for determining the LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD₅₀/ED₅₀. A CD40-binding agent (e.g., an anti-CD40 antibody)that exhibits a large therapeutic index is preferred. Where aCD40-binding agent exhibits toxic side effects, a delivery system thattargets the CD40-binding agent to the site of affected tissue can beused to minimize potential damage non-CD40-expressing cells and,thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofthe CD40 binding agent typically lies within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any CD40 bindingagent used in the method, the therapeutically effective dose can beestimated initially from cell culture assays. A dose can be formulatedin animal models to achieve a circulating plasma concentration rangethat includes the IC₅₀ (i.e., the concentration of the test compoundthat achieves a half-maximal inhibition of symptoms) as determined incell culture. Such information can be used to more accurately determineuseful doses in humans. Levels in plasma can be measured, for example,by high performance liquid chromatography.

Generally, the dosage of an anti-CD40 antibody or CD40 binding agentadministered to a patient with an immunological disorder orCD40-expressing cancer is typically about 0.1 mg/kg to about 100 mg/kgof the subject's body weight. The dosage administered to a subject isabout 0.1 mg/kg to about 50 mg/kg, about 1 mg/kg to about 30 mg/kg,about 1 mg/kg to about 20 mg/kg, about 1 mg/kg to about 15 mg/kg, orabout 1 mg/kg to about 10 mg/kg of the subject's body weight.

Exemplary doses include, but are not limited to, from 1 ng/kg to 100mg/kg. In some embodiments, a dose is about 0.5 mg/kg, about 1 mg/kg,about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg,about 11 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15mg/kg or about 16 mg/kg. The dose can be administered, for example,daily, once per week (weekly), twice per week, thrice per week, fourtimes per week, five times per week, six times per week, biweekly ormonthly. In specific embodiments, the dose is about 0.5 mg/kg/week,about 1 mg/kg/week, about 2 mg/kg/week, about 3 mg/kg/week, about 4mg/kg/week, about 5 mg/kg/week, about 6 mg/kg/week, about 7 mg/kg/week,about 8 mg/kg/week, about 9 mg/kg/week, about 10 mg/kg/week, about 11mg/kg/week, about 12 mg/kg/week, about 13 mg/kg/week, about 14mg/kg/week, about 15 mg/kg/week or about 16 mg/kg/week. In someembodiments, the dose ranges from about 1 mg/kg/week to about 15mg/kg/week.

In some embodiments, the pharmaceutical compositions comprising the CD40binding agent can further comprise a therapeutic agent, eitherconjugated or unconjugated to the binding agent. The anti-CD40 antibodyor CD40 binding agent can be co-administered in combination with one ormore therapeutic agents for the treatment or prevention of immunologicaldisorders or CD40-expressing cancers. For example, combination therapycan include a cytostatic, cytotoxic, or immunosuppressive agent.Combination therapy can also include, e.g., administration of an agentthat targets a receptor or receptor complex other than CD40 on thesurface of activated lymphocytes, dendritic cells or CD40-expressingcancer cells. An example of such an agent includes a second, non-CD40antibody that binds to a molecule at the surface of an activatedlymphocyte, dendritic cell or CD40-expressing cancer cell. Anotherexample includes a ligand that targets such a receptor or receptorcomplex. Typically, such an antibody or ligand binds to a cell surfacereceptor on activated lymphocytes, dendritic cell or CD40-expressingcancer cell and enhances the cytotoxic or cytostatic effect of theanti-CD40 antibody by delivering a cytostatic or cytotoxic signal to theactivated lymphocyte, dendritic cell or CD40-expressing cancer cell.

Such combinatorial administration can have an additive or synergisticeffect on disease parameters (e.g., severity of a symptom, the number ofsymptoms, or frequency of relapse).

With respect to therapeutic regimens for combinatorial administration,in a specific embodiment, an anti-CD40 antibody or CD40 binding agent isadministered concurrently with a therapeutic agent. In another specificembodiment, the therapeutic agent is administered prior or subsequent toadministration of the anti-CD40 antibody or CD40 binding agent, by atleast an hour and up to several months, for example at least an hour,five hours, 12 hours, a day, a week, a month, or three months, prior orsubsequent to administration of the anti-CD40 antibody or CD40 bindingagent.

Useful classes of cytotoxic or immunosuppressive agents include, forexample, antitubulin agents, auristatins (e.g., MMAE, or MMAF), DNAminor groove binders, DNA replication inhibitors, alkylating agents(e.g., platinum complexes such as cis-platin, mono(platinum),bis(platinum) and tri-nuclear platinum complexes and carboplatin),anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapysensitizers, duocarmycins, etoposides, fluorinated pyrimidines,ionophores, lexitropsins, nitrosoureas, platinols, pre-formingcompounds, purine antimetabolites, puromycins, radiation sensitizers,steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or thelike.

Individual cytotoxic or immunosuppressive agents include, for example,an androgen, anthramycin (AMC), asparaginase, 5-azacytidine,azathioprine, bleomycin, busulfan, buthionine sulfoximine, camptothecin,carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin,colchicine, cyclophosphamide, cytarabine, cytidine arabinoside,cytochalasin B, dacarbazine, dactinomycin (formerly actinomycin),daunorubicin, decarbazine, docetaxel, doxorubicin, an estrogen,5-fluordeoxyuridine, 5-fluorouracil, gramicidin D, hydroxyurea,idarubicin, ifosfamide, irinotecan, lomustine (CCNU), mechlorethamine,melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C,mitoxantrone, nitroimidazole, paclitaxel, plicamycin, procarbizine,streptozotocin, tenoposide, 6-thioguanine, thioTEPA, topotecan,vinblastine, vincristine, vinorelbine, VP-16 and VM-26.

In some typical embodiments, the therapeutic agent is a cytotoxic agent.Suitable cytotoxic agents include, for example, dolastatins (e.g.,auristatin E, AFP, MMAF, MMAE, AEB or AEVB), DNA minor groove binders(e.g., enediynes and lexitropsins), duocarmycins, taxanes (e.g.,paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38,topotecan, morpholino-doxorubicin, rhizoxin,cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin,epothilone A and B, estramustine, cryptophysins, cemadotin,maytansinoids, discodermolide, eleutherobin, or mitoxantrone.

In some embodiments, the cytotoxic agent is a conventionalchemotherapeutic such as, for example, doxorubicin, paclitaxel,melphalan, vinca alkaloids, methotrexate, mitomycin C or etoposide. Inaddition, potent agents such as CC-1065 analogues, calicheamicin,maytansine, analogues of dolastatin 10, rhizoxin, and palytoxin can belinked to the anti-CD40 antibodies or agents thereof.

In specific embodiments, the cytotoxic or cytostatic agent is auristatinE (also known in the art as dolastatin-10) or a derivative thereof.Typically, the auristatin E derivative is, e.g., an ester formed betweenauristatin E and a keto acid. For example, auristatin E can be reactedwith paraacetyl benzoic acid or benzoylvaleric acid to produce AEB andAEVB, respectively. Other typical auristatin derivatives include AFP,MMAF, and MMAE. The synthesis and structure of auristatin E and itsderivatives are described in, for example, U.S. Patent ApplicationPublication Nos. 2004-0157782 A1 and 2005-0238649; International PatentApplication No. PCT/US03/24209, International Patent Application No.PCT/US02/13435, and U.S. Pat. Nos. 6,884,869; 6,323,315; 6,239,104;6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902;5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036;5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414;the disclosures of which are incorporated by reference herein.

In specific embodiments, the cytotoxic agent is a DNA minor groovebinding agent. (See, e.g., U.S. Pat. No. 6,130,237.) For example, insome embodiments, the minor groove binding agent is a CBI compound. Inother embodiments, the minor groove binding agent is an enediyne (e.g.,calicheamicin).

Examples of anti-tubulin agents include, but are not limited to, taxanes(e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik), vincaalkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine),and dolastatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB). Otherantitubulin agents include, for example, baccatin derivatives, taxaneanalogs (e.g., epothilone A and B), nocodazole, colchicine and colcimid,estramustine, cryptophysins, cemadotin, maytansinoids, combretastatins,discodermolide, and eleutherobin.

In some embodiments, the cytotoxic agent is a maytansinoid, anothergroup of anti-tubulin agents. For example, in specific embodiments, themaytansinoid is maytansine or DM-1 (ImmunoGen, Inc.; see also Chari etal., 1992, Cancer Res. 52:127-131).

In some embodiments, the therapeutic agent is not a radioisotope.

In some embodiments, the cytotoxic or immunosuppressive agent is anantimetabolite. The antimetabolite can be, for example, a purineantagonist (e.g., azothioprine or mycophenolate mofetil), adihydrofolate reductase inhibitor (e.g., methotrexate), acyclovir,gangcyclovir, zidovudine, vidarabine, ribavarin, azidothymidine,cytidine arabinoside, amantadine, dideoxyuridine, iododeoxyuridine,poscarnet, or trifluridine.

In other embodiments, the cytotoxic or immunosuppressive agent istacrolimus, cyclosporine or rapamycin. In further embodiments, thecytotoxic agent is aldesleukin, alemtuzumab, alitretinoin, allopurinol,altretamine, amifostine, anastrozole, arsenic trioxide, bexarotene,bexarotene, calusterone, capecitabine, celecoxib, cladribine,Darbepoetin alfa, Denileukin diftitox, dexrazoxane, dromostanolonepropionate, epirubicin, Epoetin alfa, estramustine, exemestane,Filgrastim, floxuridine, fludarabine, fulvestrant, gemcitabine,gemtuzumab ozogamicin, goserelin, idarubicin, ifosfamide, imatinibmesylate, Interferon alfa-2a, irinotecan, letrozole, leucovorin,levamisole, meclorethamine or nitrogen mustard, megestrol, mesna,methotrexate, methoxsalen, mitomycin C, mitotane, nandrolonephenpropionate, oprelvekin, oxaliplatin, pamidronate, pegademase,pegaspargase, pegfilgrastim, pentostatin, pipobroman, plicamycin,porfimer sodium, procarbazine, quinacrine, rasburicase, revlimid,Sargramostim, streptozocin, tamoxifen, temozolomide, teniposide,testolactone, thioguanine, toremifene, Tositumomab, Trastuzumab,tretinoin, uracil mustard, valrubicin, vinblastine, vincristine,vinorelbine and zoledronate.

In additional embodiments, the drug is a humanized anti-HER2 monoclonalantibody; RITUXAN (rituximab; Genentech, Inc., South San Francisco,Calif.); a chimeric anti-CD20 monoclonal antibody); OVAREX (AltaRexCorporation, MA); PANOREX (Glaxo Wellcome, N.C.; a murine IgG2aantibody); Cetuximab Erbitux (Imclone Systems Inc., NY; an anti-EGFR IgGchimeric antibody); Vitaxin (MedImmune, Inc., MD); Campath I/H(Leukosite, MA; a humanized IgG1 antibody); Smart MI95 (Protein DesignLabs, Inc., CA; a humanized anti-CD33 IgG antibody); LymphoCide(Immunomedics, Inc., NJ; a humanized anti-CD22 IgG antibody); Smart ID10(Protein Design Labs, Inc., CA; a humanized anti-HLA-DR antibody);Oncolym (Techniclone, Inc., CA; a radiolabeled murine anti-HLA-Dr10antibody); Allomune (BioTransplant, CA; a humanized anti-CD2 mAb);Avastin (Genentech, Inc., CA; an anti-VEGF humanized antibody);Epratuzamab (Immunomedics, Inc., NJ and Amgen, CA; an anti-CD22antibody); and CEAcide (Immunomedics, NJ; a humanized anti-CEAantibody).

Other suitable antibodies include, but are not limited to, antibodiesagainst the following antigens: CA125, CA15-3, CA19-9, L6, Lewis Y,Lewis X, alpha fetoprotein, CA 242, placental alkaline phosphatase,prostate specific antigen, prostatic acid phosphatase, epidermal growthfactor, MAGE-1, MAGE-2, MAGE-3, MAGE-4, anti transferrin receptor, p97,MUC1-KLH, CEA, gp100, MART1, Prostate Specific Antigen, IL-2 receptor,CD20, CD52, CD33, CD22, human chorionic gonadotropin, CD38, mucin, P21,MPG, and Neu oncogene product.

In some embodiments, the therapeutic agent is an immunosuppressiveagent. The immunosuppressive agent can be, for example, gancyclovir,etanercept, tacrolimus, cyclosporine, rapamycin, cyclophosphamide,azathioprine, mycophenolate mofetil or methotrexate. Alternatively, theimmunosuppressive agent can be, for example, a glucocorticoid (e.g.,cortisol or aldosterone) or a glucocorticoid analogue (e.g., prednisoneor dexamethasone).

Suitable cyclooxygenase inhibitors include meclofenamic acid, mefenamicacid, carprofen, diclofenac, diflunisal, fenbufen, fenoprofen,ibuprofen, indomethacin, ketoprofen, nabumetone, naproxen, sulindac,tenoxicam, tolmetin, and acetylsalicylic acid.

Suitable lipoxygenase inhibitors include redox inhibitors (e.g.,catechol butane derivatives, nordihydroguaiaretic acid (NDGA),masoprocol, phenidone, lanopalen, indazolinones, naphazatrom,benzofuranol, alkylhydroxylamine), and non-redox inhibitors (e.g.,hydroxythiazoles, methoxyalkylthiazoles, benzopyrans and derivativesthereof, methoxytetrahydropyran, boswellic acids and acetylatedderivatives of boswellic acids, and quinolinemethoxyphenylacetic acidssubstituted with cycloalkyl radicals), and precursors of redoxinhibitors.

Other suitable lipoxygenase inhibitors include antioxidants (e.g.,phenols, propyl gallate, flavonoids and/or naturally occurringsubstrates containing flavonoids, hydroxylated derivatives of theflavones, flavonol, dihydroquercetin, luteolin, galangin, orobol,derivatives of chalcone, 4,2′,4′-trihydroxychalcone, ortho-aminophenols,N-hydroxyureas, benzofuranols, ebselen and species that increase theactivity of the reducing selenoenzymes), iron chelating agents (e.g.,hydroxamic acids and derivatives thereof, N-hydroxyureas,2-benzyl-1-naphthol, catechols, hydroxylamines, carnosol trolox C,catechol, naphthol, sulfasalazine, zyleuton, 5-hydroxyanthranilic acidand 4-(omega-arylalkyl)phenylalkanoic acids), imidazole-containingcompounds (e.g., ketoconazole and itraconazole), phenothiazines, andbenzopyran derivatives.

Yet other suitable lipoxygenase inhibitors include inhibitors ofeicosanoids (e.g., octadecatetraenoic, eicosatetraenoic,docosapentaenoic, eicosahexaenoic and docosahexaenoic acids and estersthereof, PGE1 (prostaglandin E1), PGA2 (prostaglandin A2), viprostol,15-monohydroxyeicosatetraenoic, 15-monohydroxy-eicosatrienoic and15-monohydroxyeicosapentaenoic acids, and leukotrienes B5, C5 and D5),compounds interfering with calcium flows, phenothiazines,diphenylbutylamines, verapamil, fuscoside, curcumin, chlorogenic acid,caffeic acid, 5,8,11,14-eicosatetrayenoic acid (ETYA),hydroxyphenylretinamide, lonapalen, esculin, diethylcarbamazine,phenantroline, baicalein, proxicromil, thioethers, diallyl sulfide anddi-(1-propenyl)sulfide.

Leukotriene receptor antagonists include calcitriol, ontazolast, BayerBay-x-1005, Ciba-Geigy CGS-25019C, ebselen, Leo Denmark ETH-615, LillyLY-293111, Ono ONO-4057, Terumo TMK-688, Boehringer Ingleheim BI-RM-270,Lilly LY 213024, Lilly LY 264086, Lilly LY 292728, Ono ONO LB457, Pfizer105696, Perdue Frederick PF 10042, Rhone-Poulenc Rorer RP 66153,SmithKline Beecham SB-201146, SmithKline Beecham SB-201993, SmithKlineBeecham SB-209247, Searle SC-53228, Sumitamo SM 15178, American HomeProducts WAY 121006, Bayer Bay-o-8276, Warner-Lambert CI-987,Warner-Lambert CI-987BPC-15LY 223982, Lilly LY 233569, Lilly LY-255283,MacroNex MNX-160, Merck and Co. MK-591, Merck and Co. MK-886, OnoONO-LB-448, Purdue Frederick PF-5901, Rhone-Poulenc Rorer RG 14893,Rhone-Poulenc Rorer RP 66364, Rhone-Poulenc Rorer RP 69698, ShionoogiS-2474, Searle SC-41930, Searle SC-50505, Searle SC-51146, SearleSC-52798, SmithKline Beecham SKandF-104493, Leo Denmark SR-2566, TanabeT-757 and Teijin TEI-1338.

Articles of Manufacture

In another aspect, an article of manufacture containing materials usefulfor the treatment of the disorders described above is included. Thearticle of manufacture comprises a container and a label. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. The containers may be formed from a variety of materials such asglass or plastic. The container holds a composition that is effectivefor treating the condition and may have a sterile access port. Forexample, the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle. The activeagent in the composition is the humanized anti-CD40 antibody. The labelon or associated with the container indicates that the composition isused for treating the condition of choice. The article of manufacturemay further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution, and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

ATCC Deposits

An ATCC deposit of monoclonal antibody S2C6 was made on May 25, 1999pursuant to the terms of the Budapest Treaty on the internationalrecognition of the deposit of microorganisms for purposes of patentprocedure. The ATCC is located at 10801 University Boulevard, Manassas,Virgina 20110-2209, USA. This ATCC deposit was given an accession numberof PTA-110. The ATCC is located at 10801 University Boulevard, Manassas,Virgina 20110-2209, USA. Any deposit is provided as a convenience tothose of skill in the art and is not an admission that a deposit isrequired under 35 U.S.C. Section 112. That described herein is not to belimited in scope by the antibody deposited, since the depositedembodiment is intended as a single illustration of certain aspects ofthe invention and any antibody that is functionally equivalent is withinthe scope of this invention. The deposit of material herein does notconstitute an admission that the written description herein contained isinadequate to enable the practice of any aspect of the invention,including the best mode thereof, nor is it to be construed as limitingthe scope of the claims to the specific illustrations that itrepresents. Indeed, various modifications of the invention in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description and fall within thescope of the appended claims.

The invention is further described in the following examples, which arenot intended to limit the scope of the invention. Cell lines describedin the following examples were maintained in culture according to theconditions specified by the American Type Culture Collection (ATCC) orDeutsche Sammlung von Mikroorganismen and Zellkulturen GmbH,Braunschweig, Germany (DMSZ). Cell culture reagents were obtained fromInvitrogen Corp. (Carlsbad, Calif.).

EXAMPLES Example 1 Production of Humanized Anti-CD40 Antibody

A humanized anti-CD40 antibody was constructed generally by importingthe CDRs of the murine anti-CD40 donor antibody into a recipient humanantibody. The donor antibody was the murine monoclonal antibody S2C6,described in U.S. Pat. No. 6,838,261, and demonstrated to providestrong, growth-promoting signals to B-lymphocytes. See, e.g., Paulie et.al., 2000, J. Immunol. 142:590. Consensus sequences for the humansubgroup III heavy chain variable domain (SEQ ID NO:2) and for the humankappa subgroup I light chain variable domain (SEQ ID NO:13) wereobtained, as generally described in Carter et. al., 1992, Proc. Natl.Acad. Sci. USA 89:4285; U.S. Pat. No. 6,037,454, and U.S. Pat. No.6,054,297 to use as the human recipient heavy and light chain domains.

The phagemid pEMX1, described in Cunningham et. al. (1989, Science243:1330-1336), contains a DNA fragment encoding the human consensusKappa-subgroup I light chain variable domain and a consensus humansubgroup III heavy chain variable domain, and is a useful vector formutagenesis as well as for expression of F(ab)s in E. coli. DNA encodingthe consensus variable domains is operably linked to an alkalinephosphatase promoter and Shine-dalgarno sequence, derived from apUC119-based plasmid, pAK2, as described in Carter et al., 1992, Proc.Natl. Acad. Sci. USA 89:4285. A unique SpeI restriction site wasengineered between the polynucleotides encoding the F(ab) light andheavy chain variable domains. pEMX1 was constructed and used inpreparing the humanized antibodies disclosed herein.

A first humanized mAb S2C6 F(ab), referred to as sgn-0, was constructedby importing the CDRs of the murine antibody into the human consensussequence framework regions by site-directed mutagenesis. Mutagenesis wasconducted generally according to the methods described in Kunkel, 1985,Proc. Natl. Acad. Sci. USA 82:488. The resulting amino acid sequences ofthe humanized F(ab) molecule (sgn-0) heavy and light chain variabledomains are shown in Table 2 (SEQ ID NO:3 and SEQ ID NO:14,respectively), and are compared with those of the donor antibody, murinemonoclonal antibody S2C6 (mMAb S2C6, also referred to herein as SGN-14;SEQ ID NO:1 and SEQ ID NO:12, respectively), and with those of the humanconsensus sequence recipient antibodies, HuV_(H) III and HuV_(L)κI (SEQID NO:2 and SEQ ID NO:13, respectively).

Plasmids were transformed into E. coli strain XL-1 Blue (Strataene, SanDiego, Calif.) for preparation of double and single stranded DNA. Eachof the light chain and heavy chain variable domains were completelysequenced using the dideoxynucleotide method (Sequenase, U.S.Biochemical Corp.). Plasmids were transformed into E. coli strain 16C9,a derivative of MM294, plated onto LB plates containing 5 μg/mlcarbenicillin, and a single colony was selected for protein expression.The 5 ml culture was added to 500 ml AP5-100 μg/ml carbenicillin andallowed to grow for 16 hours in a 4 L baffled shake flask at 37° C. APSmedia consisted of 1.5 g glucose, 11.0 g Hycase SF, 0.6 g yeast extract(certified), 0.19 g MgSO₄ (anhydrous), 1.07 g NH₄Cl, 3.73 g KCl, 1.2 gNaCl, 120 ml 1M triethanolamine, pH 7.4, to 1 L water and then sterilefiltered through 0.1 μm Sealkeen filter.

Cells were harvested by centrifugation in a 1 L centrifuge bottle(Nalgene) at 3000×g, and the supernatant removed. After freezing for 1hour, the pellet was resuspended in 25 ml cold 10 mM MES, 10 mM EDTA, pH5.0 (buffer A). 250 μl of 0.1 M PMSF (Sigma) was added to inhibitproteolysis and 3.5 ml of stock 10 mg/ml hen egg white lysozyme (Sigma)was added to aid lysis of the bacterial cell wall. After gentle shakingon ice for 1 hour, the sample was centrifuged at 40,000×g for 15minutes. The supernatant was brought to 50 ml with buffer A and loadedonto a 2 ml DEAE column equilibrated with buffer A. The flow-through wasthen applied to a protein G-Sepharose CL-4B column (Pharmacia) (0.5 mlbed volume) equilibrated with buffer A. The column was washed with 10 mlbuffer A and eluted with 3 ml 0.3 M glycine, pH 3.0, into 1.25 ml of 1MTRIS, pH8.0. The F(ab) was then buffer exchanged into PBS using aCentricon-30 filter (Amicon) and concentrated to a final volume of 0.5ml. SDS PAGE gels of the F(ab) were run to ascertain purity and themolecular weight was verified by electrospray mass spectrometry.

The humanized antibody, sgn-0, demonstrated a significantly diminishedbinding affinity for CD40 immobilized on microtiter plates, as comparedwith that of the parent, murine antibody.

TABLE 2 Variable Heavy        10         20         30        40          50 SGN-14EVQLQQSGPD LVKPGASVKI SCKASGYSFT GYYIHWVKQS HGKSLEWIGR sgn-0EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYYIHWVRQA PGKGLEWVAR HumVHIIIEVQLVESGGG LVQPGGSLRL SCAASGFTFS SYAMSWVRQA PGKGLEWVAV                             CDR-H1        60         70         80         90        100 SGN-14VIPNNGGTSY NQKFKGKAIL TVDKSSSTAY MELRSLTSED SAVYYCAREG sgn-0VIPNNGGTSY NQKFKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAREG HumVHIIIISGDGGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGR     CDR-H2       110 SGN-14 I---YWWGHG TTLTVS sgn-0 I---YWWGQG TLVTVS HumVHIIIGGGSDYWGQG TLVTVS CDR-H3 Variable Light        10         20         30         40         50 SGN-14DVVVTQTPLS LPVSLGAQAS ISCRSSQSLV HSNGNTFLHW YLQKPGQSPK sgn-0DIQMTQSPSS LSASVGDRVT ITCRSSQSLV HSNGNTFLHW YQQKPGKAPK HumKIDIQMTQSPSS LSASVGDRVT ITCRASQSIS NYL-----AW YQQKPGKAPK                            CDR-L1        60         70         80         90        100 SGN-14LLIYTVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFCSQTTHVP sgn-0LLIYTVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCSQTTHVP HumKILLIYAASSLE SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCQQYNSLP      CDR-L2                                    CDR-L3        110 SGN-14WTFGGGTKLE IQR sgn-0 WTFGQGTKVE IKR HumKI WTFGQGTKVE IKR

Example 2 Preparation of Humanized Anti-CD40 Variant Antibodies

A series of mutations were made to the template humanized antibody,sgn-0, prepared as described for Example 1. Specific mutations were madeto the DNA encoding the light and heavy chain variable domains of sgn-0by site directed mutagenesis, and the sequences of the variants producedfrom the template molecule are listed below in Tables 3 and 4.

Antibodies constructed using these variant light and heavy chainvariable domains were analyzed for binding activity. Each antibody wasdiluted to equivalent concentrations, and then serially diluted. Thediluted antibodies were assayed for binding to CD40 immobilized onmicroas say plates. Affinity binding data for the variant antibodies areshown below in Table 5. Antibodies showing binding activity approachingthat of the parent murine antibody were sgn-14, sgn-18, sgn-19, sgn-22,sgn-23, sgn-26, and sgn-27, with variants sgn-14, sgn-18, sgn-26, andsgn-27 more closely approaching that of the parent murine antibody,SGN-14, and variant sgn-26 showing the best performance in these assays.

TABLE 3 Heavy Chain Variable Domain Ab/SEQ ID NO        10         20         30         40         50 sgn-0/3EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYYIHWVRQA PGKGLEWVAR sgn-1/4EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYYIHWVRQA PGKGLEWVAR sgn-2/5EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYYIHWVRQA PGKGLEWVAR sgn-4/6EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYYIHWVRQA PGKGLEWVAR sgn-14/7EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYYIHWVRQA PGKGLEWVAR sgn-15/8EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYYIHWVRQA PGKGLEWVAR sgn-16/9EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYYIHWVRQA PGKGLEWVAR sgn-17/6EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYYIHWVRQA PGKGLEWVAR sgn-18/6EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYYIHWVRQA PGKGLEWVAR sgn-19/7EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYYIHWVRQA PGKGLEWVAR sgn-22/10EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYYIHWVRQA PGKGLEWVAR sgn-23/11EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYYIHWVRQA PGKGLEWVAR sgn-26/10EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYYIHWVRQA PGKGLEWVAR sgn-27/11EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYYIHWVRQA PGKGLEWVAR                              CDR-H1        60         70         80         90        100 sgn-0/3VIPNNGGTSY NQKFKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAREG sgn-1/4VIPNNGGTSY NQKFKGRFTI SVDNSKNTLY LQMNSLRAED TAVYYCAREG sgn-2/5VIPNNGGTSY NQKFKGRFTI SRDKSKNTLY LQMNSLRAED TAVYYCAREG sgn-4/6VIPNNGGTSY NQKFKGRATL SVDNSKNTAY LQMNSLRAED TAVYYCAREG sgn-14/7VIPNNGGTSY NQKFKGRFTL SVDNSKNTAY LQMNSLRAED TAVYYCAREG sgn-15/8VIPNNGGTSY NQKFKGRATI SVDNSKNTAY LQMNSLRAED TAVYYCAREG sgn-16/9VIPNNGGTSY NQKFKGRATL SVDNSKNTLY LQMNSLRAED TAVYYCAREG sgn-17/6VIPNNGGTSY NQKFKGRATL SVDNSKNTAY LQMNSLRAED TAVYYCAREG sgn-18/6VIPNNGGTSY NQKFKGRATL SVDNSKNTAY LQMNSLRAED TAVYYCAREG sgn-19/7VIPNNGGTSY NQKFKGRFTL SVDNSKNTAY LQMNSLRAED TAVYYCAREG sgn-22/10VIPNAGGTSY NQKFKGRFTL SVDNSKNTAY LQMNSLRAED TAVYYCAREG sgn-23/11VIPNQGGTSY NQKFKGRFTL SVDNSKNTAY LQMNSLRAED TAVYYCAREG sgn-26/10VIPNAGGTSY NQKFKGRFTL SVDNSKNTAY LQMNSLRAED TAVYYCAREG sgn-27/11VIPNQGGTSY NQKFKGRFTL SVDNSKNTAY LQMNSLRAED TAVYYCAREG     CDR-H2           110 sgn-0/3 I--- YWWGQGTLV TVS sgn-1/4 I--- YWWGQGTLV TVSsgn-2/5 I--- YWWGQGTLV TVS sgn-4/6 I--- YWWGQGTLV TVS sgn-14/7I--- YWWGQGTLV TVS sgn-15/8 I--- YWWGQGTLV TVS sgn-16/9I--- YWWGQGTLV TVS sgn-17/6 I--- YWWGQGTLV TVS sgn-18/6I--- YWWGQGTLV TVS sgn-19/7 I--- YWWGQGTLV TVS sgn-22/10I--- YWWGQGTLV TVS sgn-23/11 I--- YWWGQGTLV TVS sgn-26/10I--- YWWGQGTLV TVS sgn-27/11 I--- YWWGQGTLV TVS CDR-H3

TABLE 4 Light Chain Variable Domain Ab/SEQ ID NO        10         20         30         40         50 sgn-0/14DIQMTQSPSS LSASVGDRVT ITCRSSQSLV HSNGNTFLHW YQQKPGKAPK sgn-1/14DIQMTQSPSS LSASVGDRVT ITCRSSQSLV HSNGNTFLHW YQQKPGKAPK sgn-2/14DIQMTQSPSS LSASVGDRVT ITCRSSQSLV HSNGNTFLHW YQQKPGKAPK sgn-4/14DIQMTQSPSS LSASVGDRVT ITCRSSQSLV HSNGNTFLHW YQQKPGKAPK sgn-14/14DIQMTQSPSS LSASVGDRVT ITCRSSQSLV HSNGNTFLHW YQQKPGKAPK sgn-15/14DIQMTQSPSS LSASVGDRVT ITCRSSQSLV HSNGNTFLHW YQQKPGKAPK sgn-16/14DIQMTQSPSS LSASVGDRVT ITCRSSQSLV HSNGNTFLHW YQQKPGKAPK sgn-17/15DVQVTQSPSS LSASVGDRVT ITCRSSQSLV HSNGNTFLHW YQQKPGKAPK sgn-18/16DIQMTQSPSS LSASVGDRVT ITCRSSQSLV HSNGNTFLHW YQQKPGKAPK sgn-19/16DIQMTQSPSS LSASVGDRVT ITCRSSQSLV HSNGNTFLHW YQQKPGKAPK sgn-22/14DIQMTQSPSS LSASVGDRVT ITCRSSQSLV HSNGNTFLHW YQQKPGKAPK sgn-23/14DIQMTQSPSS LSASVGDRVT ITCRSSQSLV HSNGNTFLHW YQQKPGKAPK sgn-26/16DIQMTQSPSS LSASVGDRVT ITCRSSQSLV HSNGNTFLHW YQQKPGKAPK sgn-27/16DIQMTQSPSS LSASVGDRVT ITCRSSQSLV HSNGNTFLHW YQQKPGKAPK                               CDR-L1        60         70         80         90        100 sgn-0/14LLIYTVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCSQTTHVP sgn-1/14LLIYTVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCSQTTHVP sgn-2/14LLIYTVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCSQTTHVP sgn-4/14LLIYTVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCSQTTHVP sgn-14/14LLIYTVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCSQTTHVP sgn-15/14LLIYTVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCSQTTHVP sgn-16/14LLIYTVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCSQTTHVP sgn-17/15LLIYTVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCSQTTHVP sgn-18/16LLIYTVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YFCSQTTHVP sgn-19/16LLIYTVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YFCSQTTHVP sgn-22/14LLIYTVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCSQTTHVP sgn-23/14LLIYTVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCSQTTHVP sgn-26/16LLIYTVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YFCSQTTHVP sgn-27/16LLIYTVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YFCSQTTHVP     CDR-L2                                     CDR-L3        110sgn-0/14 WTFGQGTKVE IKR sgn-1/14 WTFGQGTKVE IKR sgn-2/14 WTFGQGTKVE IKRsgn-4/14 WTFGQGTKVE IKR sgn-14/14 WTFGQGTKVE IKR sgn-15/14WTFGQGTKVE IKR sgn-16/14 WTFGQGTKVE IKR sgn-17/15 WTFGQGTKVE IKRsgn-18/16 WTFGQGTKVE IKR sgn-19/16 WTFGQGTKVE IKR sgn-22/14WTFGQGTKVE IKR sgn-23/14 WTFGQGTKVE IKR sgn-26/16 WTFGQGTKVE IKRsgn-27/16 WTFGQGTKVE IKR

TABLE 5 Heavy Chain Light Chain Binding Binding Binding AntibodyVariable Domain Variable Domain Data 1 Data 2 Data 3 SGN-14 SEQ ID NO: 1SEQ ID NO: 12 1.00 1.33 1.16 Donor Donor hu sgn-0 SEQ ID NO: 3 SEQ IDNO: 14 75.31 75.31 — Template Template hu sgn-1 SEQ ID NO: 4 SEQ ID NO:14 23.63 19.31 — R72V Template hu sgn-2 SEQ ID NO: 5 SEQ ID NO: 14471.37 370.87 — N74K Template hu sgn-4 SEQ ID NO: 6 SEQ ID NO: 14 2.232.46 — F68A I70L R72V L79A Template 1.83 3.12 1.41 hu sgn-14 SEQ ID NO:7 SEQ ID NO: 14 1/11 0.69 — I70L R72V L79A Template 0.86 0.77 0.77 husgn-15 SEQ ID NO: 8 SEQ ID NO: 14 10.54 2.71 — F68A R72V L79A Template4.67 hu sgn-16 SEQ ID NO: 9 SEQ ID NO: 14 44.00 1.82 — F68A I69L R72VTemplate 9.78 1.83 7.99 hu sgn-17 SEQ ID NO: 6 SEQ ID NO: 15 3.60 3.56 —F68A I70L R72V L79A I2V M4V 1.76 hu sgn-18 SEQ ID NO: 6 SEQ ID NO: 160.96 1.03 F68A I70L R72V L79A Y92F 0.67 hu sgn-19 SEQ ID NO: 7 SEQ IDNO: 16 0.481 0.501 — I70L R72V L79A Y92F 1.06 0.98 0.92 1.14 hu sgn-22SEQ ID NO: 10 SEQ ID NO: 14 1.44 0.84 — N55A I70L R72V L79A Template1.41 1.80 0.93 hu sgn-23 SEQ ID NO: 11 SEQ ID NO: 14 2.11 0.90 — N55QI70L R72V L79A Template 1.58 2.38 0.72 hu sgn-26 SEQ ID NO: 10 SEQ IDNO: 16 0.92 0.92 — N55A I70L R72V L79A Y92F 1.02 1.02 1.06 hu sgn-27 SEQID NO: 11 SEQ ID NO: 16 1.04 1.40 — N55Q I70L R72V L79A Y92F 1.03 0.940.83

Example 3 In Vitro Cytotoxic Activity of Humanized Anti-CD40 Antibody

CD40⁺ and CD138′ human multiple myeloma cell lines, MM.1S, which isdexamethasone sensitive, and MM.1R, which is dexamethasone resistant, aswell as freshly isolated tumor cells (CD40⁺, CD138⁺⁺) from two multiplemyeloma patients, were treated with increasing concentrations (0-100μg/ml) of humanized S2C6 antibody for 48 hr. DNA synthesis was measuredby ³[H]-thymidine uptake. The results indicated that humanized S2C6antibody did not stimulate proliferation of MM.1S, MM.1R, or the tumorcells from the two patients (p>0.1).

To further define the cytotoxic effect of humanized S2C6 antibodyagainst these cells, cultures of MM.1S and MM.1R were treated for 6 hrwith humanized S2C6 antibody (10 μg/ml) and then cocultured with the denovo protein synthesis inhibitor cycloheximide (0.2 μg/ml) for anadditional 48 hr. Cell viability was assayed using the reduction3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) asthe indicator. Treatment with humanized S2C6 antibody and cycloheximidetriggered 50-60% cell killing in both cell lines, whereas treatment withisotype control Ig alone, with or without cycloheximide, did not inducecytotoxicity. The humanized S2C6 antibody triggered 20-30% cellcytotoxicity in the two patient tumor cell cultures, which wassignificantly enhanced in the presence of cycloheximide at a nontoxicdose (0.2 μg/ml).

Example 4 Anti-Tumor Activity of Humanized Anti-CD40 Antibody

The anti-tumor activity of the humanized anti-CD40 antibody was assayedin a SCID mouse lymphoma xenograph model. Five million Ramos tumor cellswere injected subcutaneously into SCID mice (10/group) thirteen daysprior to starting drug treatment. Murine anti-CD40 antibody or thehumanized S2C6 was given intra-peritoneally 3 times per week (4mg/kg/dose) with 8 or 5 doses administered. Mice were examined for tumorgrowth, and tumor volume was measured weekly during the 14-day studyperiod. The results in FIG. 2 show a nearly 9-fold increase in thegrowth of tumors in control mice, whereas over the same time period,tumor growth in mice treated with either murine anti-CD40 antibody orhumanized S2C6 was negligible. The data demonstrate that the humanizedantibody was as effective as the murine anti-CD40 antibody insuppressing tumor growth in this B lymphoma xenograph model.

Example 5 Prolonged Survival by Humanized Anti-CD40 Antibody

The efficacy of the humanized anti-CD40 antibody on survival oftumor-bearing mice was assayed in a SCID mouse lymphoma xenograph model.SCID mice (10/group) were inoculated intravenously with one millionRamos tumor cells three days prior to antibody treatment. Mice weretreated with murine or humanized anti-CD40 antibody, or an Ig control,adminstered intraperitoneally two times per week (4 mg/kg/dose) for atotal of five doses. The mouse cages were examined daily for mortality.The results shown in FIG. 3 show that none of the mice treated with acontrol antibody survived beyond day 34 post tumor inoculation, whereaseight of the ten mice treated with murine anti-CD40 antibody and all tenmice treated with the humanized anti-CD40 antibody remained alive ateven 90 days after tumor implant. The data demonstrate that thehumanized antibody was as effective as the murine anti-CD40 antibody inprolonging survival of SCID mice in this B lymphoma xenograph model.

Various references, including patent applications, patents, andscientific publications, are cited herein, the disclosures of which areincorporated herein by reference in their entireties. Citation oridentification of any reference herein shall not be construed as anadmission that such reference is available as prior art to the presentinvention.

The application of the teachings disclosed herein is not to be limitedin scope by the specific embodiments described herein. Indeed, variousmodifications will be within the capabilities of one having ordinaryskill in the art in light of the teachings contained herein andaccompanying examples. Such modifications are intended to fall withinthe scope of the appended claims.

1-49. (canceled)
 50. An isolated antibody or antigen-binding fragmentthat specifically binds to human CD40, comprising a humanized heavychain variable domain and a humanized light chain variable domain,wherein the heavy chain variable domain and the light chain variabledomain comprise the amino acid sequences of SEQ ID NO:10 and SEQ IDNO:16, respectively, and wherein the antibody or antigen-bindingfragment is conjugated to chemotherapeutic agent.
 51. The antibody orantigen-binding fragment of claim 50, wherein the chemotherapeutic agentis an auristatin.
 52. The antibody or antigen-binding fragment of claim51, wherein the auristatin is MMAE or MMAF.
 53. The antibody orantigen-binding fragment of claim 50, further comprising a human IgGconstant region.
 54. The antibody or antigen-binding fragment of claim53, wherein the isotype of the IgG constant region is IgG1, IgG2, IgG3,or IgG4.
 55. The antibody or antigen-binding fragment of claim 54,wherein the isotype of the IgG constant region is IgG1.
 56. The antibodyor antigen-binding fragment of claim 50, further comprising a lightchain constant domain.
 57. The antibody or antigen-binding fragment ofclaim 56, wherein the light chain constant domain is a kappa constantdomain.
 58. The antibody of claim 50, wherein the antibody furthercomprises a human immunoglobulin constant region.
 59. The antibody ofclaim 58, comprising the heavy chain and the light chain amino acidsequences set forth in SEQ ID NO:19 and SEQ ID NO:22, respectively. 60.The antibody or antigen-binding fragment of claim 50, which is anantigen-binding antibody fragment.
 61. The antigen-binding fragment ofclaim 60, wherein the antibody fragment is a Fab, a Fab′, a F(ab)₂, a Fvfragment, a diabody, a single-chain antibody, an scFv fragment or anscFv-Fc.
 62. A method for treating a subject having a CD40-expressingcancer, comprising administering to the subject the antibody orantigen-binding fragment of claim
 50. 63. The method of claim 62,wherein the cells of the CD40-expressing cancer are B lymphoblastoidcells, pancreatic cells, lung cells, breast cells, ovarian cells, coloncells, prostate cells, skin cells, head and neck cells, bladder cells,bone cells or kidney cells.
 64. The method of claim 62, wherein theCD40-expressing cancer is chronic lymphocytic leukemia, Burkitt'slymphoma, multiple myeloma, a T cell lymphoma, Non-Hodgkin's Lymphoma,Hodgkin's Disease, Waldenstrom's macroglobulinemia or Kaposi's sarcoma.65. A pharmaceutical composition comprising: (i) the antibody orantigen-binding fragment of claim 50; and (ii) a pharmaceuticallyacceptable excipient.